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  • 1995-1999  (9,003)
  • 1965-1969  (702)
  • Biochemistry and Biotechnology  (5,644)
  • Analytical Chemistry and Spectroscopy  (2,605)
  • Engineering General  (1,455)
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Material
Years
Year
Keywords
  • 101
    Electronic Resource
    Electronic Resource
    Supercritical Fluid Extraction and Supercritical Fluid Chromatography of Vitamin E in Pharmaceutical Preparations (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 300-302 
    Details
    ISSN: 0935-6304
    Keywords: Supercritical-fluid-extraction ; supercritical-fluid-chromatography ; vitamin E ; pharmaceuticals ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: No abstract
    Additional Material: 1 Ill.
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    Paper (German National Licenses)
  • 102
    Electronic Resource
    Electronic Resource
    Teicoplanin-Based Enantiomeric Separations in CZE Using a Partial Filling Technique (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 315-321 
    Details
    ISSN: 0935-6304
    Keywords: Glycopeptide antibiotics ; chiral selectors ; Teicoplanin ; enantioseparations ; CZE ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Baseline separation of the enantiomers of a number of negatively charged amino and mandelic acid derivatives was achieved in less than 10 min by capillary electrophoresis in a polyacrylamide coated capillary, using the “partial filling method” (PFM) with submillimolar concentration of Teicoplanin (TE) as the chiral selector. The influence of the charge and concentration of TE, electrolyte solution composition and pH, on the enantioresolution was examined. Further proofs were brought to corroborate the hypothesis that the enantiorecognition takes place at the D-Ala-D-Ala binding site, whose blockade is responsible for the antibacterial activity of glycopeptide antibiotics. While the dependence of the chiral recognition capabilities of TE on electrolyte solution composition and pH could limit its applicability, improved sensitivity, reduction of TE wall adsorption, resulting in a good efficiency, and high cost reduction, due to the very small amount of chiral selector required, were shown as advantages of the PFM adopted in this study.
    Additional Material: 6 Ill.
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  • 103
    Electronic Resource
    Electronic Resource
    Solvent Trapping during Large Volume Injection with an Early Vapor Exit. Part 3: The Main Cause of Volatile Component Loss during Partially Concurrent Evaporation (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 327-334 
    Details
    ISSN: 0935-6304
    Keywords: Large volume on-column injection ; partially concurrent solvent evaporation ; solvent trapping ; retaining precolumn ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---When 0.53 mm i.d. uncoated precolumns connected to a solvent vapor exit are used for sample introduction with partially concurrent solvent evaporation, substantial losses of volatile solutes are often observed. They were found to be the consequence of solute accumulation at the front end of the flooded zone, which in turn is the result of a strong pressure drop over the flooded zone owing to the formation of plugs of sample liquid. The pressure drop causes significant solvent evaporation at the front, which enriches the solute material there and causes its loss. The use of 0.32 mm i.d. restrictions between the uncoated precolumn and the vapor exit greatly reduced this problem.
    Additional Material: 10 Ill.
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  • 104
    Electronic Resource
    Electronic Resource
    Separation of Chlorophylls and Their Degradation Products using Packed Column Supercritical Fluid Chromatography (SFC) (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 339-342 
    Details
    ISSN: 0935-6304
    Keywords: Chlorophyll ; SFC ; supercritical fluid chromatography ; vegetables ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Complex mixtures of chlorophyll degradation products may arise during processing and storage of vegetable oil and green plant materials like broccoli and spinach. Determination of these compounds is important in the area of food chemistry. Therefore a method using packed column supercritical fluid chromatography (SFC) has been developed. The method comprises chromatography using a simple gradient of methanol in carbon dioxide at constant column back pressure of 30 MPa and a column temperature of 40°C. Effects of pressure and mobile phase composition showed the importance of applying a modifier gradient for optimal separation of the chlorophyll products. The method permits separation of 15 chlorophyll derivatives including chlorophyll a and b, pheophytins, and pyropheophytins on a C18 column in about 20 minutes. Identifications of the individual peaks were based on reference compounds, the retention order of the compounds, and their absorption spectra.
    Additional Material: 5 Ill.
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  • 105
    Electronic Resource
    Electronic Resource
    HRGC/FID and HRGC/MSD Analysis of the Secondary Metabolites Obtained by Different Extraction Methods from Lepechinia schiedeana, and in Vitro Evaluation of Its Antioxidant Activity (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 343-349 
    Details
    ISSN: 0935-6304
    Keywords: Capillary gas chromatography ; gas chromatography-mass spectrometry ; essential oil analysis ; Lepechinia schiedeana ; antioxidant activity ; lipid peroxidation ; Ledol; Ledol HMQC ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Steam distillation (SD), simultaneous distillation-solvent extraction (SDE), microwave-assisted solvent extraction (MWE), and supercritical (CO2) extraction (SFE) were used to isolate secondary metabolites from Lepechinia schiedeana. The various extracts were analyzed by capillary gas-chromatography, on poly (dimethylsiloxane) (DB-1) and poly(ethyleneglycol) (INNOWAX), 60 m columns, using FID or MSD (EI, 70 eV). Kováts indexes, mass spectra, or standard compounds were employed for compound identification. 43, 61, 67, and 79 compounds at concentrations above 0.01% were detected in the SD, SDE, MWE, and SFE extracts, respectively. Ledol, C15H26O, was the major constituent (20.04-36.87%) in all extracts. Oxygenated sesquiterpenes (24.36-43.14%), C10H16, monoterpenes (27.70-39.87%), and C15H24, sesquiterpenes (10.04-22.22%) were the main groups of compounds present in SD, SDE, MWE, and SFE extracts. Heavy hydrocarbons (Cn 〉 15), diterpenoids, and phytosterols were found only in MWE and SFE extracts. The antioxidant activity of Lepechinia schiedeana was measured by the HRGC quantification of the volatile carbonyl compounds, final products of lipoxidation, released in a model lipid system (sunflower oil) by the effect of the Fenton reagent. The concentration of volatile carbonyl compounds decreased by 65% when lipid oxidation was induced in the presence of macerated Lepechinia plant. The protection of polyunsaturated acids in sunflower oil was also studied by measuring their concentrations after heating of the oil (180°C, 2 h) with and without macerated Lepechinia plant.
    Additional Material: 4 Ill.
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  • 106
    Electronic Resource
    Electronic Resource
    Use of Isopropanol as a Modifier in a Hexane-Acetonitrile Based Mobile Phase for the Silver Ion HPLC Separation of Positional Isomers of Triacylglycerols Containing Long Chain Polyunsaturated Fatty Acids (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 357-361 
    Details
    ISSN: 0935-6304
    Keywords: Silver ion HPLC ; long chain polyunsaturated fatty acid ; isopropanol ; separation of positional isomers ; degree of unsaturation ; normal phase retention effect ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---A high resolution approach to silver ion HPLC was studied for the separation of positional isomers of triacylglycerols (TAGs) containing long chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA) in enzymatically synthesized structured TAGs. Isopropanol was used as a novel modifier in a hexane-acetonitrile based mobile phase for silver ion HPLC. Peak identification was based on HPLC-mass spectroscopy and selectivities of lipases. Positional isomers of TAGs containing one molecule of EPA, DHA, or DPA with saturated fatty acids (FAs) such as caprylic acid and palmitic acid were separated within 13 min using a gradient of hexane-isopropanol-acetonitrile as mobile phase. TAGs containing two or more EPA, DHA, or DPA were also separated from each other within 25 min, but their positional isomers were unresolved. The retention characteristics of the TAG were found to be related to the number of carbon atoms in the FAs present in addition to the number of double bonds and their isomeric configuration. One isomer with an unsaturated FA in the sn-2 position eluted faster than the other with the unsaturated FA in the sn-1 or 3 position. Species with longer chain FAs attached to TAGs with the same degree of unsaturation eluted faster than those that have shorter chain FAs. For example, docosapentaenoylhexadecanoyloctanoin (DPA/C16/C8) was eluted faster than dioctanoyldocosapentaenoin (DPA/C8/C8).
    Additional Material: 2 Ill.
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  • 107
    Electronic Resource
    Electronic Resource
    A Computer Method for Predicting the Electrophoretic Mobility of Peptides (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 373-378 
    Details
    ISSN: 0935-6304
    Keywords: Electrophoresis ; electrophoretic mobilities ; CZE ; MEKC ; peptides ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---A computer model is presented for the prediction of the electrophoretic mobilities of peptides from physical constants derived from their amino acid sequences. The model assumes that the electrophoretic mobility can be represented by a product of four functions according to the relation:  lcomp= l(L)w(W)q(Q)c(CC), where L (a length parameter) is represented by the number of amino acid residues of the peptide, W (a width parameter) is represented by the average residue mass, Q = the charge of the peptide, and CC = the position of the center of charge relative to the center of mass. The model was used to calculate the electrophoretic mobilities of peptides in a 50 mM phosphate buffer at pH 2.5. Sixty-four test peptides ranging in size from 2 to 39 amino acid residues were used for this study. The calculated mobilities show excellent correlation with experimental measurements with a correlation coefficient greater than 0.98.
    Additional Material: 5 Tab.
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  • 108
    Electronic Resource
    Electronic Resource
    Simultaneous Measurement of Hydrocarbons and Sulfur Compounds using Flame Ionization and Sulfur Chemiluminescence Detection for Sulfur Simulated Distillation (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 386-390 
    Details
    ISSN: 0935-6304
    Keywords: Sulfur chemiluminescence detection ; sulfur simulated distillation ; boiling point distribution ; simultaneous FID/SCD analysis ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A gas chromatographic system for the simultaneous acquisition of hydrocarbon and sulfur chromatograms was developed. Detection of sulfur compounds is achieved using a sulfur chemiluminescence detector (SCD) mounted in series with a flame ionization detector (FID). A constant fraction of the effluent of the FID is transferred to the SCD by means of a fixed restrictor. Unlike previous versions of this approach, the FID is not used to generate the chemiluminescent sulfur species. Rather, the FID is operated under optimum conditions for hydrocarbon analysis and a furnace is used to generate the chemiluminescent sulfur species. The system permits dual acquisition of the hydrocarbon and sulfur signals in a single analysis with a single column, since the detectors are operated in a serial fashion. The application of sulfur simulated distillation using this approach was examined, since this requires simultaneous universal and sulfur selective detection. Precision of absolute response of both the FID and SCD was typically less than 2% RSD for a standard reference material.
    Additional Material: 3 Ill.
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  • 109
    Electronic Resource
    Electronic Resource
    A Novel Interface for Optimized Coupling of Gas Chromatography and Supersonic Jet Spectroscopy (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 391-394 
    Details
    ISSN: 0935-6304
    Keywords: REMPI-TOFMS ; laser mass spectrometry ; supersonic jet ; GC-MS ; interface ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---The laser-based methods Laser Induced Fluorescence (LIF) and Resonance-Enhanced Multi-Photon Ionization (REMPI) can be used as highly selective detection modes for gas chromatography (GC). One major prerequisite for successful application of these detection methods is the availability of appropriate and reliable interfaces between the GC effluent and the molecular beam inlet. When a pulsed supersonic molecular beam (jet) source is used, the analyte molecules are efficiently cooled, allowing maximum selectivity of the laser spectroscopic detection methods. However, several technical problems have to be solved for practical realization of a GC-supersonic jet valve hyphenation. The pulsed jet interface should not interfere with the GC properties and the supersonic molecular beam properties. Further a good working cycle for the conversion from the continuously flowing GC current to the pulsed jet gas flow should be attained. This paper presents a novel setup of a GC-pulsed jet interface. The construction allows temporal and spatial compression of the analyte molecules in jet gas pulse and thus an increase of the detection sensitivity. Moreover, the GC effluent comes into contact only with glass surfaces and not with valve parts like plungers and seals. This reduces memory effects and sample decomposition. The valve setup is tested with a REMPI-TOFMS instrument.
    Additional Material: 2 Ill.
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  • 110
    Electronic Resource
    Electronic Resource
    Selective Determination of Se4+ and Se6+ Using SPME and GC/MS (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 414-416 
    Details
    ISSN: 0935-6304
    Keywords: Selenium ; water ; SPME ; GC/MS ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---A method for the selective determination of Se4+ and Se6+ using solid phase microextraction (SPME) and GC/MS analysis is presented. Se4+ is selectively derivatized by reaction with 4,5-dichloro-1,2-phenylenediamine to form the corresponding piazselenolo complex, extracted by the SPME fiber, and determined by GC/MS. The RSD at a 5 μg/L concentration was 9.88% and the theoretical detection limit 6 ng/L. The method was employed to test real matrices; tap and river water were analyzed before and after spiking giving a recovery rate of 102% in river water and 97% in tap water.
    Additional Material: 3 Ill.
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  • 111
    Electronic Resource
    Electronic Resource
    Determination of Vapor Liquid Equilibrium from the Kovats Retention Index on Dimethylsilicone using the Wilson Mixing Model (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 565-588 
    Details
    ISSN: 0935-6304
    Keywords: Gas chromatography ; Kovats retention index ; vapor pressure ; boiling point ; Wilson activity coefficient ; binary phase diagrams ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Non-ideal mixing in a dimethylsilicone stationary phase is modeled according to the Wilson activity coefficient. Pure liquid vapor pressures of alkylated compound series are calculated from capillary GLC retention with the functional group heat of solution in a polymer solvent. The new method uses the Kovats index, molar mass, and functional group to determine the bubble line of a compound. Boiling points at reduced and normal pressure are compared to literature values of 194 gasoline components. An unlike molecular pair interaction parameter is derived, using only bubble line data of the pure liquids. Binary phase diagrams are constructed and compared to vapor liquid equilibrium data.
    Additional Material: 22 Ill.
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  • 112
    Electronic Resource
    Electronic Resource
    A Strategy for the Simplification and Solution of Complex Chromatographic Analysis Problems Utilizing Two-Dimensional Mapping of Retention Indexes Followed by Computer Modeling of Heart Cuts from Serially Coupled Columns Containing Different Stationary Phases (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 589-593 
    Details
    ISSN: 0935-6304
    Keywords: Computer modeling of GC ; computer simulation of GC ; computer-assisted GC analysis ; GC multicolumn analysis ; heart cutting GC analysis ; computer-assisted column design ; computer-assisted selectivity tuning ; retention index mapping ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---The analysis of four components in a mixture containing many possible interferents is simplified by heart-cutting serially connected columns containing stationary phases of widely different polarities. The design of the proper lengths of the two columns and timing the heart-cuts is accomplished with the aid of a computer simulation of the problem. The computer program calculates the proper lengths of column to produce the shortest analysis time and also outputs the predicted chromatograms from both columns. The chromatogram from the first column is used to construct the timing gates for the heart-cuts and the chromatogram from the second or analytical column is used to set integration gates for the selected compounds of interest.
    Additional Material: 7 Ill.
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  • 113
    Electronic Resource
    Electronic Resource
    Indirect Fluorimetric Detection of Cyclodextrins via Postcolumn Mixing with 2-p-Toluidinyl-6-naphthalenesulfonate in Liquid Chromatography (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 609-612 
    Details
    ISSN: 0935-6304
    Keywords: Cyclodextrins ; 2-p-toluidinyl-6-naphthalenesulfonate ; indirect fluorimetric detection ; liquid chromatography ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Cyclodextrins (CD's) were visualized by postcolumn mixing with 2-p-toluidinylnaphthalene sulfonate (TNS) in liquid chromatography. The indirect detection is based on fluorescence enhancement due to the formation of an inclusion complex between TNS and CD's. β-CD gave larger signal intensity than α- and γ-CD. Different selectivities were observed for alkyl-bonded silica stationary phases with different chain length.
    Additional Material: 7 Ill.
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  • 114
    Electronic Resource
    Electronic Resource
    Analysis of Methylcitric Acid by Enantioselective Multidimensional Gas Chromatography-Mass Spectrometry (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 604-608 
    Details
    ISSN: 0935-6304
    Keywords: Enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC-MS) ; methylcitric acid (MCA) ; inherited metabolic disease ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Methylcitric acid (2-hydroxybutane-1,2,3-tricarboxylic acid-MCA) is a structural analogue of citric acid, but due to an additional methyl group it is a chiral molecule with two stereogenic centers and thus four stereoisomers are conceivable. MCA occurs naturally as prominent metabolite in body fluids of patients with inherited metabolic diseases such as propionic acidemia, methylmalonic aciduria, or holocarboxylase synthetase deficiency. Therefore methylcitric acid is considered to be an important diagnostic marker for these diseases. MCA is most likely produced from accumulated propionyl-CoA in these diseases by the enzyme si-citrate synthase from the citric acid cycle; however, there are other enzymes known which could catalyze the same reaction with different stereoselectivity, such as re-citrate synthase or the more specific enzyme methylcitrate synthase, found in microorganisms. Almost all methods dealing with MCA in the literature are non-enantioselective. For that reason there is no information about occurrence of MCA enantiomers in healthy people, patients with propionic acidemia, methylmalonic aciduria, or holocarboxylase synthetase deficiency and about value of enantiomeric distribution for diagnosis and long-term treatment. The enantioselective analysis of MCA as corresponding trimethyl ester was achieved by enantioselective multidimensional gas chromatography coupled with mass spectrometry using heptakis-(2,3-di-O-methyl-6-O-tert-butyl-dimethylsilyl)-β-cyclodextrin as chiral stationary phase. The described method allows a reliable screening of MCA in complex matrices like urine without time consuming sample preparation and with mass selective detection. During this investigation urine samples from various patients and healthy controls were analyzed. As concluded, MCA is a good diagnostic marker and can be easily measured by the method presented. Only the two stereoisomers (2S,3R) and (2S,3S) were detectable in patients and healthy controls. The varying ratios of these stereoisomers cannot presently be correlated with the health status of patients, although there are some indications that this might be possible. However, the quantitative levels of MCA, determined as the ratio of MCA absolute peak area divided by 1,000 to the creatinine contents of urine samples in this investigation, showed a dependence on the state of health and MCA would thus also be a possible marker for long-term treatment. Such a substance is of major interest nowadays since there are different studies searching for such a long-term marker in propionic acidemia or methylmalonic aciduria.
    Additional Material: 4 Ill.
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  • 115
    Electronic Resource
    Electronic Resource
    Relative Recoveries of Hydrocarbons from Geological Samples Using High Temperature Supercritical Fluid Extraction: Contributions from Thermal Desorption (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 623-627 
    Details
    ISSN: 0935-6304
    Keywords: Supercritical fluid extraction ; hydrocarbons ; high temperature ; thermal desorption ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The extraction of n-alkanes, polyaromatic hydrocarbons (naphthalene, methylated naphthalenes, phenanthrene, methylated phenanthrenes, anthracene, and methylated anthracenes) and biomarkers (hopanes) from Posidonia shale by high temperature supercritical fluid (HT-SFE) carbon dioxide extraction has been evaluated, including the relative contributions from thermal desorption and relative comparisons to conventional Soxhlet extraction. These current results confirm those of previous studies indicating a widely differing extractability of strongly and weakly associated hydrocarbons in ancient sediments and the significantly higher recoveries possible using HT-SFE compared to conventional Soxhlet extraction. The present study also demonstrates that these high HT-SFE recoveries are due to true extraction rather than simple thermal desorption and that this procedure is a useful tool to study speciation as well as total extractable hydrocarbons from sediments.
    Additional Material: 2 Ill.
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  • 116
    Electronic Resource
    Electronic Resource
    Stereoisomeric Flavor Compounds LXXXII Dill Ether and Its Stereoisomers: Synthesis and Enantioselective Analysis (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 631-634 
    Details
    ISSN: 0935-6304
    Keywords: Dill ether ; stereoselective synthesis ; dill ether stereoisomers ; enantioselective multidimensional gas chromatography-mass spectrometry (enantio-MDGC/MS) ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---No abstract
    Additional Material: 2 Ill.
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  • 117
    Electronic Resource
    Electronic Resource
    Complete Separation of Isomeric Penta- and Hexachloronaphthalenes by Capillary Gas Chromatography (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 639-643 
    Details
    ISSN: 0935-6304
    Keywords: Cyclodextrin ; Halowax ; hexachloronaphthalene ; PCN ; polychlorinated naphthalenes ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---No abstract
    Additional Material: 3 Ill.
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  • 118
    Electronic Resource
    Electronic Resource
    Theory of Fast Capillary Gas Chromatography Part 4: Column Performance vs.Liquid Film Thickness (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 501-508 
    Details
    ISSN: 0935-6304
    Keywords: Fast GC ; film inefficiency factor ; high pressure drop ; thick film columns ; thin film columns ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The negative effect of the liquid stationary phase film thickness on the column efficiency is strongest for peaks with retention factors, k, in the vicinity of k = 0.3-0.4 and rapidly diminishes with the departure of k from that region. Additionally, at the high pressure drop required for fast analysis of complex mixtures, the negative effect of the same film thickness diminishes with the increase in column length, regardless of the k values. In practice, it is recommended to ignore the film thickness and optimize the columns as thin film ones regardless of their actual film thickness. Accounting for the film thickness results only in a modest improvement in the resolution for a few affected peaks - those with k = 0.3-0.4. However, this improvement comes at the cost of a substantial increase in analysis time, and should be used only as the action of the last resort.
    Additional Material: 4 Ill.
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  • 119
    Electronic Resource
    Electronic Resource
    Isolation and Characterization of Wax Esters in Fennel and Caraway Seed Oils by SPE-GC (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 514-520 
    Details
    ISSN: 0935-6304
    Keywords: Solid-phase extraction ; high temperature - gas chromatography ; gas chromatography - mass spectrometry ; wax esters fennel seed oil ; caraway seed oil ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---A rapid method for the isolation and quantitative determination of wax esters in vegetable oils was developed. For the first time wax esters in oils were separated from the triglyceride matrix by means of solid-phase extraction, which allows rapid sample preparation in parallel and therefore a high sample throughput. The thus obtained wax ester fractions of fennel and caraway seed oils were analyzed by high temperature gas chromatography. GC-MS analyses were carried out using electron impact ionization in order to characterize the wax ester fraction. With respect to the results of the GC-MS analyses different isomers of saturated wax esters with the same carbon number were observed. Additional monounsaturated wax esters with an unsaturated fatty acid moiety were identified.
    Additional Material: 4 Ill.
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  • 120
    Electronic Resource
    Electronic Resource
    Comparative Methodology for Isolation of Flavonoid Glycosides from Clusia criuva Cambess (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 527-530 
    Details
    ISSN: 0935-6304
    Keywords: Clusia criuva Cambess ; counter-current chromatography ; flavonoids ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---The family Guttiferae belongs to the superorder Theiflorae, order Theales and according to Dahlgren's system of classification, it is equivalent to Hypericaceae (=Clusiaceae) [1]. This family consists of 49 genera encompassing over 1000 species of wide distribution in the tropics as shrubs and trees [2]. Plants of the genus Clusia have been used in folk medicine for the treatment of different kind of illness such as: febrifuge, anti rheumatic, purgative, for stomach problems and, in Brazil, they are very commonly used to heal wounds [3]. The key of any study of material from natural sources is the availability of suitable separation methods for the isolation of pure products [4-6]. Tentatives for separations using chomatography on solid support phases were not very efficients in the fractionation of Clusia criuva Cambess methanolic/ethyl acetate extracts. However, the use of counter-current chromatography, an all liquid technique, showed advantages in comparison with the more traditional liquid-solid separation methods and even with HPLC. In this sense it was possible to isolate flavonoid glycosides from ethyl acetate extract of Clusia criuva, in a very short operation time.
    Additional Material: 3 Ill.
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    Fast Solvating Gas Chromatography of Environmentally Important Compounds Using Polymer-Encapsulated Silica Particles (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 541-546 
    Details
    ISSN: 0935-6304
    Keywords: Solvating gas chromatography ; gas chromatography ; fast separation ; pesticides ; herbicides ; aldehydes ; packed capillary columns ; polymer-encapsulated silica ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Rapid separations of selected environmentally important polar compounds using polymer-encapsulated silica stationary phases and a carbon dioxide mobile phase under solvating gas chromatography (SGC) conditions are reported. Ten underivatized short chain aldehydes and ten nitrogen-containing herbicides were separated within 1 min and 5 min, respectively, using a 30 cm×250 μm i. d. column packed with diol-bonded, polyethylenimine (PEI)-coated, and hexamethyldisilazane (HMDS)-end-capped silica particles (5 μm, 120 Å). Seven organophosphorus pesticides were resolved in less than 5 min using a 30 cm×250 μm i. d. column packed with polymethylhydrosiloxane-deactivated and SE-54 encapsulated silica particles. Separation numbers per unit time increased with pressure and temperature ramps. Both rapid pressure and temperature programming can be used to increase the speed of SGC. The effects of pressure and temperature on apparent retention factors of solutes with various polarities were investigated using diol-PEI-HMDS silica particles in SGC.
    Additional Material: 9 Ill.
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    Evaluation of Dead Time Calculation in Reversed-Phase Liquid Chromatography Using a Multiparametric Mathematical Method (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 559-564 
    Details
    ISSN: 0935-6304
    Keywords: Liquid chromatography ; dead time ; multiparametric least-squares adjustment ; homologous series ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---In reversed phase liquid chromatography (RP-HPLC), the validity of a multiparametric non-linear least-squares regression iterative method for the determination of the column dead time tM and the regression parameter (slope b), based on the use of alkan-2-ones, alkyl aryl ketones, and 1-nitroalkanes has been evaluated. The determination of tM and b has been extensively studied for two mobile phase systems acetonitrile-water and methanol-water on seven octadecyl-C18 and one octyl C8 analytical columns. The calculated tM and b values were compared with those obtained by Guardino's and Grobler's methods. The influence exerted thereon by the nature of the homologous series, the mobile phase composition, and the packing materials were investigated.
    Additional Material: 5 Tab.
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    Supercritical Fluid Extraction (SFE) Optimization by Full-Factorial Design for the Determination of Irganox 1076, Irgafos 168, and Chimassorb 81 in Virgin and Recycled Polyolefins (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 553-558 
    Details
    ISSN: 0935-6304
    Keywords: SFE ; experimental design ; additives ; recycling ; polyolefins ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---The performance and feasibility of supercritical fluid extraction (SFE) applied to the extraction of some antioxidants (Irganox 1076, Irgafos 168) and one UV-stabilizer (Chimassorb 81) from both virgin and recycled low density polyethylene (LDPE), and virgin high density polyethylene (HDPE) are studied. Due to the high number of variables a full-factorial design has been applied to minimize the number of experiments required to reach the optimum extraction conditions. Further analysis has been carried out off-line by reversed-phase HPLC. Modification of the physical properties of the polymeric matrix and increased number of recycling cycles as well as the influence of physical properties on the efficiency of SFE are also discussed.
    Additional Material: 1 Ill.
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  • 124
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    Capillary Electrophoresis as an Efficient Tool in the Determination of Low Association Constants between Polyhydroxy Compound and Samarium Cation (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 595-598 
    Details
    ISSN: 0935-6304
    Keywords: Xylitol ; samarium ; weak association ; equilibrium constant ; capillary electrophoresis ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---No abstract
    Additional Material: 1 Ill.
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  • 125
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    Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 599-603 
    Details
    ISSN: 0935-6304
    Keywords: Capillary electrochromatography ; retention ; mixed stationary phases ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.
    Additional Material: 5 Ill.
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  • 126
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    Comparison of Retention Indices of Some Explosives and Related Compounds Calculated by Different Mathematical Methods in Reversed-Phase Liquid Chromatography (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 613-618 
    Details
    ISSN: 0935-6304
    Keywords: Liquid chromatography ; retention index ; multiparametric least-squares adjustment ; homologous series ; nitramines ; nitroaromatics ; aminoaromatics and nitrophenols ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---In reversed phase liquid chromatography, the retention indices of some neutral and acidic explosives and related compounds (nitramines, nitroaromatics, aminoaromatics, and nitrophenols) based on the alkan-2-one, alkyl aryl ketone, and 1-nitroalkane retention index standards have been determined by the application of a new mathematical adaptation method, viz. a multiparametric least-squares regression iterative method. This method was applied to two types of columns. The first group includes six octadecyl-C18 columns with different packing materials and obtained from different manufacturers, while the second group comprises one octyl-C8 column. The retention indices have been extensively studied using either methanol-water or methanol-phosphate buffer mobile phase systems. The calculated multiparametric retention indices values were compared with those obtained by Guardino's, Grobler's, and Kovàts' methods. The influences of the concentration of the organic modifier in the mobile phase, aqueous mobile phase pH, and the column packing material on the retention indices of the explosives were also investigated. Good agreement was observed between the retention indices calculated by the use of the four mathematical methods for both neutral and acidic explosives.
    Additional Material: 4 Tab.
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    Extraction and Clean-Up Method for the Determination of Twenty Organochlorine Pesticide Residues in Tomatoes by GLC-ECD (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 619-622 
    Details
    ISSN: 0935-6304
    Keywords: Organochlorine compounds ; pesticides ; food analysis ; tomatoes ; GLC-ECD ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---A method is described for the simultaneous extraction and determination of twenty organochlorine pesticides residues [hexachlorocyclohexane (HCH) isomers (α, β, γ, δ), aldrin, dieldrin, endrin, heptachlor and its epoxide (HE), hexachlorobenzene (HCB), α-endosulfan, o,p′-DDD, p,p′-DDD, o,p′-DDE, p,p′-DDE, o,p′-DDT, p,p′-DDT, dicofol, methoxychlor, and mirex] that were spiked in tomatoes. Samples were extracted with ethyl acetate, and the extract was subjected to a rapid clean-up using a Florisil column. The residues were determined by GLC-ECD using two columns of different polarity. The calibration graph correlation coefficient ranged from 0.9903 to 0.9990. The recoveries ranged from 95 to 99% with relative standard deviation ranging from 0.5 to 14.7% in the concentration range 0.005 to 0.2 μg/g. The limits of detection ranged from 0.004 to 0.05 μg/g and the limits of quantification ranged from 0.005 to 0.174 μg/g.
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    Total p-Nitrophenol Determination in Urine Samples of Subjects Exposed to Parathion and Methyl-Parathion by SPME and GC/MS (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 628-630 
    Details
    ISSN: 0935-6304
    Keywords: p-Nitrophenol ; urine ; SPME ; GC/MS ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---No abstract
    Additional Material: 2 Ill.
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    A Rapid Method for Fingerprinting the Organic Fraction in Archeological Samples Using a PTV as a Thermodesorber (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 635-638 
    Details
    ISSN: 0935-6304
    Keywords: High resolution gas chromatography ; thermal desorption ; archeological cosmetic powders ; PTV injector ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---No abstract
    Additional Material: 5 Ill.
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    Properties and Applications of Mixed Packing Capillary Electrochromatography (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 666-670 
    Details
    ISSN: 0935-6304
    Keywords: Capillary electrochromatography ; mixed packing technique ; electroosmotic flow ; retention mechanism ; fast analysis ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Mixed packing capillary electrochromatography (MP CEC) with the stationary phase comprising a physical mixture of strong cation exchange (SCX) phase and octadecysilyl (ODS) phase was developed. With the existence of a sulfonic acid group on the surface of SCX, not only could the electroosmotic flow (EOF) remain high at low pH, but also the hydrophilicity of the stationary phase was increased greatly, leading to broad adaptable ranges of both pH and organic modifier concentration in the mobile phase. At the same time, with the coexistence of C18 on the surface of ODS, both the retention and the resolution of samples were improved. Accordingly, MP CEC combined the advantages of both SCX and ODS columns. Effects of operation parameters on EOF and the capacity factors of solutes as well as the retention mechanism of such a column were studied systematically. In addition, MP CEC columns were used in the analysis of strong polar solutes as well as for the high speed separation of acidic, basic, and neutral compounds in a single run.
    Additional Material: 7 Ill.
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    Cyclocholates as Chiral Selectors for Capillary Gas Chromatography - Effect of Temperature Conditioning on the Chromatographic Behavior of the Stationary Phase (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 671-678 
    Details
    ISSN: 0935-6304
    Keywords: Enantioselective capillary gas chromatography ; chiral stationary phase ; chiral recognition mechanism ; cholic acid ; cyclocholates ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---The suitability of cyclocholates as chiral selectors in gas chromatography has been evaluated. We present the synthesis and characterization of two cyclocholates, viz. 3α,7α-diacetoxycyclo[3]cholate and 3α,7α-diacetoxycylo[4]cholate. Mixtures of these new selectors with polysiloxanes were tested as chiral stationary phases in capillary gas chromatography. Several enantiomer separations of common racemates were achieved with the 3α,7α-diacetoxycyclo[3]cholate at 10% in OV-1701 (w/w). It was shown that column efficiency was strongly dependent on temperature and that enantioselectivity was very sensitive to column conditioning. This chromatographic behavior suggested that cyclocholates were only dispersed in polysiloxane. Thus, it was assumed that chiral discrimination occurred via enantioselective adsorption interactions of enantiomers at the surface of the solid chiral selector dispersed in the polysiloxane matrix OV-1701.
    Additional Material: 11 Ill.
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    Comparative Study of Packed and Capillary Column Chromatography for the Characterization of Vacuum Gas Oils (1999)
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 22 (1999), S. 683-686 
    Details
    ISSN: 0935-6304
    Keywords: Vacuum gas oils ; packed column chromatography ; n-paraffins ; carbon number distribution ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: ---Gas chromatographic methods are of widespread use in the petroleum industry for assessing petroleum and derivatives quality. The feedstocks for catcracking processes are very sensitive to the n-paraffin contents, which can be accurately determined using capillary column chromatography. Nevertheless, packed column chromatography does have potential advantages, viz. sometimes shorter analysis times, lower costs, higher column capacity, for vacuum gas oil analysis. This paper presents a method for such a determination using a Dexsil-300 packed column. The results obtained (n-alkane contents, carbon number distribution) agree fairly well with those obtained on a 30 m × 0.25 mm DB-1 fused silica capillary column.
    Additional Material: 3 Ill.
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    Mixing in Stars and the Evolution of the 3He Abundance (1998)
    Springer
    Space science reviews 84 (1998), S. 199-206 
    Details
    ISSN: 1572-9672
    Keywords: Nuclear reactions ; Nucleosynthesis ; Abundances ; Stars:Evolution ; Interior ; Rotation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract We first recall the observational and theoretical facts that constitute the so-called 3He problem. We then review the chemical anomalies that could be related to the destruction of 3He in red giants stars. We show how a simple consistent mechanism can lead to the destruction of 3He in low mass stars and simultaneously account for the low 12C/13C ratios and low lithium abundances observed in giant stars of different populations. This process should both naturally account for the recent measurements of 3He/H in galactic HII regions and allow for high values of 3He observed in some planetary nebulae. We propose a simple statistical estimation of the fraction of stars that may be affected by this process.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005027519798
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  • 134
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    Improved protein refolding using hollow-fibre membrane dialysis (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 590-599 
    Details
    ISSN: 0006-3592
    Keywords: protein refolding ; hollow-fibre membrane ; dialysis ; carbonic anhydrase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have used a cellulose acetate, hollow-fibre (HF) ultrafiltration membrane to refold bovine carbonic anhydrase, loaded into the lumen space, by removing the denaturant through controlled dialysis via the shell side space. When challenged with GdnHCl-denatured carbonic anhydrase, 70% of the loaded protein reptated through the membrane into the circulating dialysis buffer. Reptation occurred because the protein, in its fully unfolded configuration, was able to pass through the pores. The loss of carbonic anhydrase through the membrane was controlled by the dialysis conditions. Dialysis against 0.05 M Tris-HCl for 30 min reduced the denaturant around the protein to a concentration that allowed the return of secondary structure, increasing the hydrodynamic radius, thus preventing protein transmission. Under these conditions a maximum of 42% of carbonic anhydrase was recovered (from a starting concentration of 5 mg/mL) with 94% activity. This is an improvement over refolding carbonic anhydrase by simple batch dilution, which gave a maximum reactivation of 85% with 35% soluble protein yield. The batch refolding of carbonic anhydrase is very sensitive to temperature; however, during HF refolding between 0 and 25°C the temperature sensitivity was considerably reduced. In order to reduce the convection forces that give rise to aggregation and promote refolding the dialyzate was slowly heated from 4 to 25°C. This slow, temperature-controlled refolding gave an improved soluble protein recovery of 55% with a reactivation yield of 90%. The effect of a number of additives on the refolding system performance were tested: the presence of PEG improved both the protein recovery and the recovered activity from the membrane, while the detergents Tween 20 and IGEPAL CA-630 increased only the refolding yield. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 590-599, 1998.
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    Metabolic engineering (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 119-120 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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    Activity losses among T4 lysozyme variants after adsorption to colloidal silica (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 658-662 
    Details
    ISSN: 0006-3592
    Keywords: T4 lysozyme ; silica nanoparticles ; synthetic enzyme variants ; surface-induced conformational change ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Maintaining a specific molecular conformation is essential for the proper functioning of an enzyme. A substantial loss of catalytic activity can occur from the displacement caused by even a single amino acid substitution. Activity may also be lost as an enzyme undergoes a conformational change during adsorption. In this study, we investigated the effect of thermostability on the activities of three T4 lysozyme variants after adsorption to 9 nm colloidal silica particles. Less-stable T4 lysozyme variants lost more activity after adsorption than did more stable variants, apparently because they experienced more extensive structural alteration. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 658-662, 1998.
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    On-line metabolic pathway analysis based on metabolic signal flow diagram (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 139-148 
    Details
    ISSN: 0006-3592
    Keywords: metabolic engineering ; pathway analysis ; metabolic and energetic model ; physiological state ; Saccharomyces cerevisiae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this work, an integrated modeling approach based on a metabolic signal flow diagram and cellular energetics was used to model the metabolic pathway analysis for the cultivation of yeast on glucose. This approach enables us to make a clear analysis of the flow direction of the carbon fluxes in the metabolic pathways as well as of the degree of activation of a particular pathway for the synthesis of biomaterials for cell growth. The analyses demonstrate that the main metabolic pathways of Saccharomyces cerevisiae change significantly during batch culture. Carbon flow direction is toward glycolysis to satisfy the increase of requirement for precursors and energy. The enzymatic activation of TCA cycle seems to always be at normal level, which may result in the overflow of ethanol due to its limited capacity. The advantage of this approach is that it adopts both virtues of the metabolic signal flow diagram and the simple network analysis method, focusing on the investigation of the flow directions of carbon fluxes and the degree of activation of a particular pathway or reaction loop. All of the variables used in the model equations were determined on-line; the information obtained from the calculated metabolic coefficients may result in a better understanding of cell physiology and help to evaluate the state of the cell culture process. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:139-148, 1998.
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    Experimental determination of group flux control coefficients in metabolic networks (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 149-153 
    Details
    ISSN: 0006-3592
    Keywords: Metabolic Control Analysis ; flux control coefficients ; top down MCA ; metabolic engineering ; Corynebacterium glutamicum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Grouping of reactions around key metabolite branch points can facilitate the study of metabolic control of complex metabolic networks. This top-down Metabolic Control Analysis is exemplified through the introduction of group (flux, as well as concentration) control coefficients whose magnitudes provide a measure of the relative impact of each reaction group on the overall network flux, as well as on the overall network stability, following enzymatic amplification. In this article, we demonstrate the application of previously developed theory to the determination of group flux control coefficients. Experimental data for the changes in metabolic fluxes obtained in response to the introduction of six different environmental perturbations are used to determine the group flux control coefficients for three reaction groups formed around the phosphoenolpyruvate/pyruvate branch point. The consistency of the obtained group flux control coefficient estimates is systematically analyzed to ensure that all necessary conditions are satisfied. The magnitudes of the determined control coefficients suggest that the control of lysine production flux in Corynebacterium glutamicum cells at a growth base state resides within the lysine biosynthetic pathway that begins with the PEP/PYR carboxylation anaplorotic pathway. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:149-153, 1998.
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    Application of mathematical tools for metabolic design of microbial ethanol production (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 154-161 
    Details
    ISSN: 0006-3592
    Keywords: central carbon pathways ; metabolic optimization ; ethanol production ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Many attempts to engineer cellular metabolism have failed due to the complexity of cellular functions. Mathematical and computational methods are needed that can organize the available experimental information, and provide insight and guidance for successful metabolic engineering. Two such methods are reviewed here. Both methods employ a (log)linear kinetic model of metabolism that is constructed based on enzyme kinetics characteristics. The first method allows the description of the dynamic responses of metabolic systems subject to spatiotemporal variations in their parameters. The second method considers the product-oriented, constrained optimization of metabolic reaction networks using mixed-integer linear programming methods. The optimization framework is used in order to identify the combinations of the metabolic characteristics of the glycolytic enzymes from yeast and bacteria that will maximize ethanol production. The methods are also applied to the design of microbial ethanol production metabolism. The results of the calculations are in qualitative agreement with experimental data presented here. Experiments and calculations suggest that, in resting Escherichia coli cells, ethanol production and glucose uptake rates can be increased by 30% and 20%, respectively, by overexpression of a deregulated pyruvate kinase, while increase in phosphofructokinase expression levels has no effect on ethanol production and glucose uptake rates. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:154-161, 1998.
    Additional Material: 7 Ill.
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    Multiple mechanisms controlling carbon metabolism in bacteria (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 170-174 
    Details
    ISSN: 0006-3592
    Keywords: catabolite repression ; phosphotransferase system ; inducer exclusion ; inducer expulsion ; protein kinase ; transcriptional regulation ; transport regulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Catabolite repression is a universal phenomenon, found in virtually all living organisms. These organisms range from the simplest bacteria to higher fungi, plants, and animals. A mechanism involving cyclic AMP and its receptor protein (CRP) in Escherichia coli was established years ago, and this mechanism has been assumed by many to serve as the prototype for catabolite repression in all organisms. However, recent studies have shown that this mechanism is restricted to enteric bacteria and their close relatives. Cyclic AMP-independent mechanisms of catabolite repression occur in other bacteria, yeast, plants, and even E. coli. In fact, single-celled organisms such as E. coli, Bacillus subtilis, and Saccharomyces cerevisiae exhibit multiple mechanisms of catabolite repression, and most of these are cyclic AMP-independent. The mechanistic features of the best of such characterized processes are briefly reviewed, and references are provided that will allow the reader to delve more deeply into these subjects. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:170-174, 1998.
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    How will bioinformatics influence metabolic engineering? (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 162-169 
    Details
    ISSN: 0006-3592
    Keywords: bioinformatics ; metabolic engineering ; genetic engineering ; mathematical analysis ; stoichiometry ; enzyme kinetics ; modal analysis ; genetic circuits ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Ten microbial genomes have been fully sequenced to date, and the sequencing of many more genomes is expected to be completed before the end of the century. The assignment of function to open reading frames (ORFs) is progressing, and for some genomes over 70% of functional assignments have been made. The majority of the assigned ORFs relate to metabolic functions. Thus, the complete genetic and biochemical functions of a number of microbial cells may be soon available. From a metabolic engineering standpoint, these developments open a new realm of possibilities. Metabolic analysis and engineering strategies can now be built on a sound genomic basis. An important question that now arises; how should these tasks be approached? Flux-balance analysis (FBA) has the potential to play an important role. It is based on the fundamental principle of mass conservation. It requires only the stoichiometric matrix, the metabolic demands, and some strain specific parameters. Importantly, no enzymatic kinetic data is required. In this article, we show how the genomically defined microbial metabolic genotypes can be analyzed by FBA. Fundamental concepts of metabolic genotype, metabolic phenotype, metabolic redundancy and robustness are defined and examples of their use given. We discuss the advantage of this approach, and how FBA is expected to find uses in the near future. FBA is likely to become an important analysis tool for genomically based approaches to metabolic engineering, strain design, and development. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:162-169, 1998.
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    Artificial promoters for metabolic optimization (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 191-195 
    Details
    ISSN: 0006-3592
    Keywords: control analysis ; Lactococcus lactis ; gene expression ; flux ; oligonucleotide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we review some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering, and examine their advantages and disadvantages in different contexts. In a recent approach, artificial promoters for modulating gene expression in micro-organisms were constructed using synthetic degenerated oligonucleotides. From this work, a promoter library was obtained for Lactococcus lactis, containing numerous individual promoters and covering a wide range of promoter activities. Importantly, the range of promoter activities was covered in small steps of activity change. Promoter libraries generated by this approach allow for optimization of gene expression and for experimental control analysis in a wide range of biological systems by choosing from the promoter library promoters giving, e.g., 25%, 50%, 200%, and 400% of the normal expression level of the gene in question. If the relevant variable (e.g., the flux or yield) is then measured with each of these constructs, then one can calculate the control coefficient and determine the optimal expression level. One advantage of the method is that the construct which is found to have the optimal expression level is then, in principle, ready for use in the industrial fermentation process; another advantage is that the system can be used to optimize the expression of different enzymes within the same cell. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:191-195, 1998.
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    Engineering protein-based machines to emulate key steps of metabolism (biological energy conversion) (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 175-190 
    Details
    ISSN: 0006-3592
    Keywords: protein-based polymers ; inverse temperature transitions ; hydrophobic-induced pKa shifts ; waters of hydrophobic hydration ; five axioms for protein engineering; microwave dielectric relaxation ; a universal mechanism for biological energy conversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society. In addition, metabolic engineering can utilize the principles, whereby proteins function, to design new protein-based machines to fulfill roles for society that proteins have never been called upon throughout evolution to fulfill.This article presents arguments for a universal mechanism whereby proteins perform their diverse energy conversions; it begins with background information, and then asserts a set of five axioms for protein folding, assembly, and function and for protein engineering. The key process is the hydrophobic folding and assembly transition exhibited by properly balanced amphiphilic protein sequences. The fundamental molecular process is the competition for hydration between hydrophobic and polar, e.g., charged, residues. This competition determines Tt, the onset temperature for the hydrophobic folding and assembly transition, Nhh, the numbers of waters of hydrophobic hydration, and the pKa of ionizable functions.Reported acid-base titrations and pH dependence of microwave dielectric relaxation data simultaneously demonstrate the interdependence of Tt, Nhh and the pKa using a series of microbially prepared protein-based poly(30mers) with one glutamic acid residue per 30mer and with an increasing number of more hydrophobic phenylalanine residues replacing valine residues. Also, reduction of nicotinamides and flavins is shown to lower Tt, i.e., to increase hydrophobicity.Furthermore, the argument is presented, and related to an extended Henderson-Hasselbalch equation, wherein reduction of nicotinamides represents an increase in hydrophobicity and resulting hydrophobic-induced pKa shifts become the basis for understanding a primary energy conversion (proton transport) process of mitochondria. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:175-190, 1998.
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    Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: Effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 248-259 
    Details
    ISSN: 0006-3592
    Keywords: Escherichia coli ; Chloramphenicol Acetyltransferase (CAT) ; Culture Redox Potential (CRP) ; Dithiothreitol (DTT) ; reducing agents ; molecular chaperones ; proteases ; heat shock ; stress response ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/μg total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/μg CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (σ32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/μg CAT min.) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 248-259, 1998.
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    A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 261-272 
    Details
    ISSN: 0006-3592
    Keywords: effective diffusive permeability ; diffusion coefficient ; biofilm ; cell density ; review ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms are reviewed. Effective diffusive permeabilities, the parameter appropriate to the analysis of reaction-diffusion interactions, depend on solute type and biofilm density. Three categories of solute physical chemistry with distinct diffusive properties were distinguished by the present analysis. In order of descending mean relative effective diffusive permeability (De/Daq) these were inorganic anions or cations (0.56), nonpolar solutes with molecular weights of 44 or less (0.43), and organic solutes of molecular weight greater than 44 (0.29). Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms as proposed by Hinson and Kocher (1996). A conceptual model of biofilm structure is proposed in which each cell is surrounded by a restricted permeability envelope. Effective diffusion coefficients, which are appropriate to the analysis of transient penetration of nonreactive solutes, are generally similar to effective diffusive permeabilities in biofilms of similar composition. In three studies that examine diffusion of very large molecular weight solutes ( 〉 5000) in biofilms, the average ratio of the relative effective diffusion coefficient of the large solute to the relative effective diffusion coefficient of either sucrose or fluorescein was 0.64, 0.61, and 0.36. It is proposed that large solutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:261-272, 1998.
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    II. Electrostatic effect in the aggregation of heat-denatured RNase A and implications for protein additive design (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 281-285 
    Details
    ISSN: 0006-3592
    Keywords: protein aggregation ; RNase A ; protein formulation ; protein additives ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the previous study (part I), heat-denatured RNase A aggregation was shown to depend on the solution pH. Interestingly, at pH 3.0, the protein did not aggregate even when exposed to 75°C for 24 h. In this study, electrostatic repulsion was shown to be responsible for the absence of aggregates at that pH. While RNase A aggregation was prevented at the extremely acidic pH, this is not an environment conducive to maintaining protein function in general. Therefore, attempts were made to confer electrostatic repulsion near neutral pH. In this study, heat-denatured RNase A was mixed with charged polymers at pH 7.8 in an attempt to provide the protein with excess surface cations or anions. At 75°C, SDS and dextran sulfate were successful in preventing RNase A aggregation, whereas their cationic, nonionic, and zwitterionic analogs did not do so. We believe that the SO3- groups present in both additives transformed the protein into polyanionic species, and this may have provided a sufficient level of electrostatic repulsion at pH 7.8 and 75°C to prevent aggregation from proceeding. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:281-285, 1998.
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    An experimental and modeling study on the removal of mono-chlorobenzene vapor in biotrickling filters (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 328-343 
    Details
    ISSN: 0006-3592
    Keywords: biotrickling filters ; biotrickling filter modeling ; mono-chlorobenzene ; biodegradation kinetics of mono-chlorobenzene ; chlorinated VOC emissions ; biofiltration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Removal of mono-chlorobenzene (m-CB) vapor from airstreams was studied in a biotrickling filter (BTF) operating under counter-current flow of the air and liquid streams. Experiments were performed under various values of inlet m-CB concentration, air and/or liquid volumetric flow rates, and pH of the recirculating liquid. Conversion of m-CB was never below 70% and at low concentrations exceeded 90%. A maximum removal rate of about 60 gm-3-reactor h-1 was observed. Conversion of m-CB was found to increase as the values of liquid and air flow rate increase and decrease, respectively. The effects of pH and frequency of medium replenishment on BTF performance were also investigated. The process was successfully described with a detailed mathematical model, which accounts for mass transfer and kinetic effects based on m-CB and oxygen availability. Solution of the model equations yielded m-CB and oxygen concentration profiles in all three phases (airstream, liquid, biofilm). It is predicted that oxygen has a controling effect on the process at high inlet m-CB concentrations. From independent, suspended culture, experiments it was found that m-CB biodegradation follows Andrews inhibitory kinetics. The kinetic constants were found to remain practically unchanged after the culture was used in BTF experiments for 8 months. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:328-343, 1998.
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    Effect of some operating variables on citrate recovery from model solutions by electrodialysis (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 344-350 
    Details
    ISSN: 0006-3592
    Keywords: electrodialysis ; citric acid ; pH ; temperature ; Faraday efficiency ; solute recovery efficiency ; specific energy consumption ; solute flux ; water flux ; feed solute concentration ; electric current density ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of pH and temperature (θ) on the overall performance indicators (i.e., solute recovery, ρ, and Faraday, η, efficiencies; specific energy consumption, ε, solute, JS, and water, JW, fluxes) of batch electrodialytic recovery of citric acid from model solutions was assessed at different values of feed solute concentration (cSf) and electric current density (j). Regardless of the initial feed concentration used, ρ and JS were found to be independent of θ; η and JW exhibited a positive trend with respect to θ, while ε a negative one. At the maximum temperature tested (33°C), as the pH of the feed solution was varied from 3 to 7, ρ increased from 0.90 ± 0.08 to 0.97 ± 0.02, η grew from 0.09 ± 0.02 to 0.50 ± 0.01, JS practically doubled, ε reduced about 8 times, but JW increased from 3 to 4 times. So, the optimal conditions for this technique are to be determined by balancing the savings in the investment and maintenance costs against the energy costs. © John Wiley & Sons, Inc. Biotechnol Bioeng 59:344-350, 1998.
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    Stability and activity modulation of chymotrypsins in AOT reversed micelles by protein-interface interaction: Interaction of α-chymotrypsin with a negative interface leads to a cooperative breakage of a salt bridge that keeps the catalytic active conformation (Ile16-Asp194) (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 360-363 
    Details
    ISSN: 0006-3592
    Keywords: chymotrypsin ; enzyme stability ; reversed micelles ; interface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The stability of α-chymotrypsin and δ-chymotrypsin was studied in reversed micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. α-Chymotrypsin is inactivated at the interface and at the water pool, while δ-chymotrypsin is inactivated only at the water pool. The mechanism of inactivation at the interface is related to the interaction of N-terminal group alanine 149 (absent in δ-chymotrypsin) with the negative interface. The dependence of enzyme activity on water content of these two enzymes in reversed micelles of AOT is also related with the interface interaction, since δ-chymotrypsin does not have a bell-shaped curve as observed for α-chymotrypsin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:360-363, 1998.
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    High-density perfusion culture of insect cells with a BioSep ultrasonic filter (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 351-359 
    Details
    ISSN: 0006-3592
    Keywords: bioreactor ; high density ; insect cells ; perfusion ; Sf9 ; ultrasonic filter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The baculovirus/insect cell expression system has provided a vital tool to produce a high level of active proteins for many applications. We have developed a very high-density insect cell perfusion process with an ultrasonic filter as a cell retention device. The separation efficiency of the filter was studied under various operating conditions. A cell density of over 30 million cells/mL was achieved in a controlled perfusion bioreactor and cell viability remained greater than 90%. Sf9 cells from a high-density culture and a spinner culture were infected with two recombinant baculoviruses expressing genes for the production of human chitinase and monocyte-colony inhibition factor. The protein yield on a cell basis from infecting high-density Sf9 cells was the same as or higher than that from the spinner Sf9 culture. Virus production from the high-density culture was similar to that from the spinner culture. The results show that the ultrasonic filter did not affect insect cells' ability to support protein expression and virus production following infection with baculovirus. The potential applications of the high-density perfusion culture for large-scale protein expression from Sf9 cells are also highlighted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:351-359, 1998.
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    Prevention of marine biofouling using a conductive paint electrode (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 374-378 
    Details
    ISSN: 0006-3592
    Keywords: conductive paint electrode ; prevention of marine biofouling ; fishing net ; alternating potential ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Conductive paint electrode was used for marine biofouling on fishing nets by electrochemical disinfection. When a potential of 1.2 V vs. a saturated calomel electrode (SCE) was applied to the conductive paint electrode, Vibrio alginolyticus cells attached on the electrode were completely killed. By applying a negative potential, the attached cells were removed from the surface of the electrode. Changes in pH and chlorine concentration were not observed at potentials in the range -0.6 ∼1.2 V vs. SCE. In a field experiment, accumulation of the bacterial cells and formation of biofilms on the electrode were prevented by application of an alternating potential, and 94% of attachment of the biofouling organisms was inhibited electrically on yarn used for fishing net coated with conductive paint. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:374-378, 1998.
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    Mass transfer studies on immobilized α-chymotrypsin biocatalysts prepared by deposition for use in organic medium (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 364-373 
    Details
    ISSN: 0006-3592
    Keywords: porous supports ; internal and external diffusion ; active site accessibility ; enzyme loading ; kinetically controlled dipeptide synthesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mass transfer limitations were studied in enzyme preparations of α-chymotrypsin made by deposition on different porous support materials such as controlled pore glasses, Celite, and polyamides of different particle sizes. It is the onset of mass transfer limitations that determines the position of the activity optimum with respect to enzyme loading on each support. The evidence of various experiments indicates that internal diffusional limitations are the important mechanism for the observed mass transfer limitations. External diffusion was not found to play an important role under the conditions used, and it was also found that when immobilizing multilayers of enzyme the buried enzyme molecules are active to a large extent. An extreme situation is observed on Celite at very high loadings. Under these conditions, this support is expected to have its pores completely filled with packed enzyme molecules, and then it is the diffusion within the enzyme layer that determines the observed rate. As the enzyme loading increases, the area of contact between the deposited enzyme layers and the liquid solution inside the pores diminishes, causing a decrease on the observed rate of an intrinsically fast reaction which apparently is incongruous with the presence of more enzyme in the system. This work shows that mass transfer limitations can be an important factor when working with immobilized enzymes in organic media, and its study should be carried out in order to avoid undesired reduced enzyme activities and specificities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:364-373, 1998.
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    Degradation of perchloroethylene and dichlorophenol by pulsed-electric discharge and bioremediation (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 438-444 
    Details
    ISSN: 0006-3592
    Keywords: bioremediation ; plasma discharge ; dichlorophenol degradation ; perchloroethylene degradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pulsed electric discharge (PED) and bioremediation were combined to create a novel two-stage system which dechlorinates the halogenated pollutants, 2,4-dichlorophenol and perchloroethylene, with repetitive (0.1-1 kHz), short pulse (∼100 ns), low voltage (40-80 kV) discharges and then mineralizes the less chlorinated products with aerobic bacteria. A 6.1 mM aqueous dichlorophenol sample was cycled through the PED reactor (60 kV of applied pulsed voltage and 300 Hz) 6 times, resulting in the release of 55% of the initial dichlorophenol chloride ions (1 mM Cl- removed each cycle). The respective average specific efficiency is 0.4-0.6 keV/(Cl- molecule). Pseudomonas mendocina KR1, which grows in minimal medium supplemented with phenol but not with dichlorophenol, increased in cell density in all cultures supplemented with the PED-treated DCP samples and yielded a maximum of two-fold additional Cl- released compared to the PED-related alone. The number of PED-treatment cycles, voltage, and frequency were also varied, showing that both cell densities and overall dichlorophenol dechlorination were highly dependent upon the number of PED-treatment cycles, rather than the tested voltages and frequencies. Using this two-stage treatment system, PED released 31% of the initial chloride ions from dichlorophenol (after three cycles at 40-45 kV and 1.2 kHz) while P. mendocina KR1 in the second stage increased dechlorination to 90%. These results were corroborated by the 35% additional chloride release found with activated sludge cultures. Perchloroethylene (0.6 mM) was similarly treated in a first-stage PED reactor (80% chloride removal after four cycles) followed by biodegradation of the dechlorinated products with a recombinant toluene o-monooxygenase-expressing Pseudomonas fluorescens strain. Gas chromatographic analysis showed that the PED reactor created less-chlorinated byproducts (i.e., trichloroethylene) that were removed (74%) upon exposure to the recombinant bacterium. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:438-444, 1998.
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    Antisense strategies for glycosylation engineering of Chinese hamster ovary (CHO) cells (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 445-450 
    Details
    ISSN: 0006-3592
    Keywords: CHO cells ; glycosylation engineering ; antisense ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:445-450, 1998.
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    Analysis of protein fouling during ultrafiltration using a two-layer membrane model (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 451-460 
    Details
    ISSN: 0006-3592
    Keywords: protein fouling ; membrane transport ; ultrafiltration ; adsorption ; filtration ; composite membrane ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein fouling can significantly alter both the flux and retention characteristics of ultrafiltration membranes. There has, however, been considerable controversy over the nature of this fouling layer. In this study, hydraulic permeability and dextran sieving data were obtained both before and after albumin adsorption and/or filtration using polyethersulfone ultrafiltration membranes. The dextran molecular weight distributions were analyzed by gel permeation chromatography to evaluate the sieving characteristics over a broad range of solute size. Protein fouling caused a significant reduction in the dextran sieving coefficients, with very different effects seen for the diffusive and convective contributions to dextran transport. The changes in dextran sieving coefficients and diffusive permeabilities were analyzed using a two-layer membrane model in which a distinct protein layer is assumed to form on the upstream surface of the membrane. The data suggest that the protein layer formed during filtration was more tightly packed than that formed by simple static adsorption. Hydrodynamic calculations indicated that the pore size of the protein layer remained relatively constant throughout the adsorption or filtration, but the thickness of this layer increased with increasing exposure time. These results provide important insights into the nature of protein fouling during ultrafiltration and its effects on membrane transport. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:451-460, 1998.
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    Contribution of protein charge to partitioning in aqueous two-phase systems (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 461-470 
    Details
    ISSN: 0006-3592
    Keywords: aqueous two-phase separation ; protein partitioning ; T4 lysozyme ; electrochemical partitioning ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein partitioning in aqueous two-phase systems based on phase-forming polymers is strongly affected by the net charge of the protein, but a thermodynamic description of the charge effects has been hindered by conflicting results. Many of the difficulties could be because of problems in isolating electrochemical effects from other interactions of phase components.We explored charge effects on protein partitioning in poly(ethylene glycol)-dextran two-phase systems by using two series of genetically engineered charge modifications of bacteriophage T4 lysozyme produced in Escherichia coli. The two series, one in the form of charged-fusion tails and the other in the form of charge-change point mutations, provided matching net charges but very different polarity. Partition coefficients of both series were obtained and interfacial potential differences of the phase systems were measured. Multi-angle laser light scattering measurements were also performed to determine second virial coefficients. A semi-empirical model accounting for the roles of both charge and non-charge effects on protein partitioning behavior is proposed, and the results predicted from the model are compared to the results from the experiments. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:461-470, 1998.
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    Ammonium ion and glucosamine dependent increases of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 518-528 
    Details
    ISSN: 0006-3592
    Keywords: ammonium ; UDP-GlcNAc ; N -glycosylation ; BHK-21 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of different ammonium concentrations and glucosamine on baby hamster kidney (BHK)-21 cell cultures grown in continuously perfused double membrane bioreactors was investigated with respect to the final carbohydrate structures of a secretory recombinant glycoprotein. The human interleukin-2 (IL-2) mutant glycoprotein variant IL-Mu6, which bears a novel N-glycosylation site (created by a single amino acid exchange of Gln100 to Asn), was produced under different defined protein-free culture conditions in the presence or absence of either glutamine, NH4Cl, or glucosamine. Recombinant glycoprotein products were purified and characterized by amino acid sequencing and carbohydrate structural analysis using matrix-assisted laser desorption ionization time of flight mass spectrometry, high-pH anion-exchange chromatography with pulsed amperometric detection, and methylation analysis. In the absence of glutamine, cells secreted glycoprotein forms with preponderantly biantennary, proximal fucosylated carbohydrate chains (85%) with a higher NeuAc content (58%). Under standard conditions in the presence of 7.5 mM glutamine, complex-type N-glycans were found to be mainly biantennary (68%) and triantennary structures (33%) with about 50% containing proximal α1-6-linked fucose; 37% of the antenna were found to be substituted with terminal α2-3-linked N-acetylneuraminic acid. In the presence of 15 mM exogenously added NH4Cl, a significant and reproducible increase in tri- and tetraantennary oligosaccharides (45% of total) was detected in the secretion product. In glutamin-free cultures supplemented with glucosamine, an intermediate amount of high antennary glycans was detected. The increase in complexity of N-linked oligosaccharides is considered to be brought about by the increased levels of intracellular uridine diphosphate-GlcNAc/GalNAc. These nucleotide sugar pools were found to be significantly elevated in the presence of high NH3/NH4+ and glucosamine concentrations. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 518-528, 1998.
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    Metabolic modeling of polyhydroxybutyrate biosynthesis (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 557-570 
    Details
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; polyhydroxyalkanoates ; metabolic engineering ; mathematical modeling ; enzyme kinetics ; regulation of metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 557-570, 1998.
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    Enhanced secretion of human granulocyte colony-stimulating factor directed by a novel hybrid fusion peptide from recombinant Saccharomyces cerevisiae at high cell concentration (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 600-609 
    Details
    ISSN: 0006-3592
    Keywords: rhG-CSF ; fusion protein ; secretion efficiency ; glycosylation ; multimer ; conformation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The synthesis and secretion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GAL1-10 upstream activating sequence (UASG), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1β. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1β fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O-glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 600-609, 1998.
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    Protein refolding by reversed micelles utilizing solid-liquid extraction technique (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 620-623 
    Details
    ISSN: 0006-3592
    Keywords: protein refolding ; reversed micelles ; solid-liquid extraction ; RNase A ; DNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 620-623, 1998.
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    Identification and control of oxidative metabolism in Saccharomyces cerevisiae during transient growth using calorimetric measurements (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 610-619 
    Details
    ISSN: 0006-3592
    Keywords: dynamic model ; Saccharomyces cerevisiae ; oxidative capacity ; feedback control ; calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this study was to characterize the dynamic adaptation of the oxidative capacity of Saccharomyces cerevisiae to an increase in the glucose supply rate and its implications for the control of a continuous culture designed to produce biomass without allowing glucose to be diverted into the reductive metabolism. Continuous cultures subjected to a sudden shift-up in the dilution rate showed that the glucose uptake rate increased immediately to the new feeding rate but that the oxygen consumption could not follow fast enough to ensure a completely oxidative metabolism. Thus, part of the glucose assimilated was degraded by the reductive metabolism, resulting in a temporary decrease of biomass concentration, even if the final dilution rate was below Dcrit. The dynamic increase of the specific oxygen consumption rate, qO2, was characterized by an initial immediate jump followed by a first-order increase to the maximum value. It could be modeled using three parameters denoted qjumpO2, qmaxO2, and a time constant τ. The values for the first two of the parameters varied considerably from one shift to another, even when they were performed under identical conditions. On the basis of this model, a time-dependent feed flow rate function was derived that should permit an increase in the dilution rate from one value to another without provoking the appearance of reductive metabolism. The idea was to increase the glucose supply in parallel with the dynamic increase of the oxidative capacity of the culture, so that all of the assimilated glucose could always be oxidized. Nevertheless, corresponding feed-profile experiments showed that deviations in the reductive metabolism could not be completely suppressed due to variability in the model parameters. Therefore, a proportional feedback controller using heat evolution rate measurements was implemented. Calorimetry provides an excellent and rapid estimate of the metabolic activity. Satisfactory control was achieved and led to constant biomass yields. Ethanol accumulated only up to 0.49 g L-1 as compared to an accumulation of 1.82 g L-1 without on-line control in the shift-up experiment to the same final dilution rate. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 610-619, 1998.
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    Establishment of an apoptosis-resistant and growth-controllable cell line by transfecting with inducible antisense c-Jun gene (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 65-72 
    Details
    ISSN: 0006-3592
    Keywords: c-jun ; cell cycle ; apoptosis ; antisense ; growth deprivation ; F-MEL ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: F-MEL cells were transfected with the c-jun antisense gene located downstream of a glucocorticoid-inducible MMTV promoter, and the obtained cells were named c-jun AS cells. When the c-jun AS cells were treated with dexamethasone (DEX) in DMEM supplemented with 10% serum, the growth of the cells was completely suppressed for a duration of 16 days with a high cell viability exceeding 86%. The c-jun expression in the c-jun AS cells was suppressed moderately in the absence of DEX and strongly in the presence of DEX. The c-jun AS cells grew well and reached a density of 106 cells/mL without supplementation of any serum components. Viability was greater than 80% after the cells had been cultured for 8 days in the absence of DEX. The c-jun AS cells stayed at a constant cell density and high viability above 80% for 8 days when they were cultured in the presence of DEX under serum deprivation. In contrast, the wild type F-MEL cells were unable to grow and died by apoptosis in 3 days under serum deprivation. Internucleosomal cleavage of DNA, a landmark of apoptosis, was clearly detectable. Thus the c-jun AS cell line that is resistant to apoptosis induced by serum deprivation and can reversibly and viably be growth-arrested was established. A dual-signal model was proposed to explain the experimental result, the interlinked regulation of apoptosis, and growth by c-jun.© 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:65-72, 1998.
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    Deactivation and conformational changes of cutinase in reverse micelles (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 380-386 
    Details
    ISSN: 0006-3592
    Keywords: reverse micelles ; cutinase ; deactivation ; conformational changes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380-386, 1998.
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    Conserved water molecules in the specificity pocket of serine proteases and the molecular mechanism of Na+ binding (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 34-42 
    Details
    ISSN: 0887-3585
    Keywords: allostery ; buried water molecules ; molecular recognition ; Na+ site ; thrombin ; trypsin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Conservation of clusters of buried water molecules is a structural motif present throughout the serine protease family. Frequently, these clusters are shaped as water channels forming extensive hydrogen-bonding networks linked to the protein backbone. The most conspicuous example is the water channel present in the specificity pocket of trypsin and thrombin. In thrombin, other vitamin K-dependent proteases, and some complement factors, Na+ binds in this water channel and enhances allosterically the catalytic activity of the enzyme, whereas digestive and fibrinolytic proteases are devoid of such regulation. A comparative analysis of proteases with and without Na+ binding capability reveals the role of the water channel in maintaining the structural organization of the specificity pocket and in Na+ coordination. This enables the formulation of a molecular mechanism for Na+ binding in thrombin and leads to the identification of the structural changes necessary to engineer a functional Na+ site and enhanced catalytic activity in trypsin and other proteases. Proteins 30:34-42, 1998. © 1998 Wiley-Liss, Inc.
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    Is the molten globule a third thermodynamic state of protein? The example of α-lactalbumin (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 43-48 
    Details
    ISSN: 0887-3585
    Keywords: molten globule ; α-lactalbumin ; calorimetry ; viscosimetry ; derivative spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Thermal and denaturant-induced transitions of the acid molten globule state of bovine α-lactalbumin (acid [A] state) are analyzed by scanning calorimetry, titration calorimetry, viscosimetry, and derivative spectroscopy. A denaturant-induced heat effect of the A state is shown by a calorimetric difference titration of the A-state versus unfolded (reduced) α-lactalbumin. However, changes of viscosity and derivative spectra do not parallel the heat effect. At thermal denaturation monitored by derivative spectroscopy and scanning microcalorimetry the presence of a gradual transition in α-lactalbumin A state is shown. The results are consistent with the existence of tertiary interactions in the A state and the absence of a cooperative unfolding transition of the molten globule. The results do not support the idea that the molten globule is a third thermodynamic state. Proteins 30:43-48, 1998. © 1998 Wiley-Liss, Inc.
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    Exploring hydrophobic sites in proteins with xenon or krypton (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 61-73 
    Details
    ISSN: 0887-3585
    Keywords: xenon ; krypton ; hydrophobic cavity ; protein-ligand binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: X-ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate-oxidase from Aspergillus flavus, mosquitocidal δ-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-α. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel-like structures. These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy-atom derivatives in the isomorphous replacement method of structure determination. Proteins 30:61-73, 1998. © 1998 Wiley-Liss, Inc.
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    Structure-based thermodynamic design of peptide ligands: Application to peptide inhibitors of the aspartic protease endothiapepsin (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 74-85 
    Details
    ISSN: 0887-3585
    Keywords: folding and binding ; kinetics ; pepstatin A ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The prediction of binding affinities from structure is a necessary requirement in the development of structure-based molecular design strategies. In this paper, a structural parameterization of the energetics previously developed in this laboratory has been incorporated into a molecular design algorithm aimed at identifying peptide conformations that minimize the Gibbs energy. This approach has been employed in the design of mutants of the aspartic protease inhibitor pepstatin A. The simplest design strategy involves mutation and/or chain length modification of the wild-type peptide inhibitor. The structural parameterization allows evaluation of the contribution of different amino acids to the Gibbs energy in the wild-type structure, and therefore the identification of potential targets for mutation in the original peptide. The structure of the wild-type complex is used as a template to generate families of conformational structures in which specific residues have been mutated. The most probable conformations of the mutated peptides are identified by systematically rotating around the side-chain and backbone torsional angles and calculating the Gibbs potential function of each conformation according to the structural parametrization. The accuracy of this approach has been tested by chemically synthesizing two different mutants of pepstatin A. In one mutant, the alanine at position five has been replaced by a phenylalanine, and in the second one a glutamate has been added at the carboxy terminus of pepstatin A. The thermodynamics of association of pepstatin A and the two mutants have been measured experimentally and the results compared with the predictions. The difference between experimental and predicted Gibbs energies for pepstatin A and the two mutants is 0.23 ± 0.06 kcal/mol. The excellent agreement between experimental and predicted values demonstrates that this approach can be used in the optimization of peptide ligands. Proteins 30:74-85, 1998. © 1998 Wiley-Liss, Inc.
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    Oxygen and proton pathways in cytochrome c oxidase (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 100-107 
    Details
    ISSN: 0887-3585
    Keywords: cytochrome c oxidase ; proton pump ; oxygen diffusion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Cytochrome c oxidase is a redox-driven proton pump, which couples the reduction of oxygen to water to the translocation of protons across the membrane. The recently solved x-ray structures of cytochrome c oxidase permit molecular dynamics simulations of the underlying transport processes. To eventually establish the proton pump mechanism, we investigate the transport of the substrates, oxygen and protons, through the enzyme.   Molecular dynamics simulations of oxygen diffusion through the protein reveal a well-defined pathway to the oxygen-binding site starting at a hydrophobic cavity near the membrane-exposed surface of subunit I, close to the interface to subunit III.   A large number of water sites are predicted within the protein, which could play an essential role for the transfer of protons in cytochrome c oxidase. The water molecules form two channels along which protons can enter from the cytoplasmic (matrix) side of the protein and reach the binuclear center. A possible pumping mechanism is proposed that involves a shuttling motion of a glutamic acid side chain, which could then transfer a proton to a propionate group of heme α3. Proteins 30:100-107, 1998. © 1998 Wiley-Liss, Inc.
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    Specific fluorescent labeling of two functional domains in RNA polymerase α subunit (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 183-192 
    Details
    ISSN: 0887-3585
    Keywords: chemical modification ; fluorescent probe ; site-directed mutagenesis ; cysteine-free protein ; alanine scanning ; enzyme reconstitution ; protein-DNA interaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A monomercury derivative of fluoresceine acetate (FMMA) was previously suggested as a specific reagent reacting with only one of four cysteine (Cys) residues in the α subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated α subunit and α subunit assembled in the holoenzyme. In both cases, the highest reactivity was identified for Cys-269 positioned in the regulatory helix of C-terminal domain (CTD) which includes the contact sites for both class-I transcription factors and DNA UP elements. Substitution of Ala for both Cys-269 and Cys-176 completely eliminates the reactivity of α subunit against the fluorescent dye, supporting the prediction that another reactive amino acid under native conformation is Cys-176, which is positioned within or near the region important for α dimerization and its binding of β' subunit. In the isolated α subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant α-subunits, bearing only one Cys residue at either 269 or 176, could be reconstituted into locally modified and active enzymes. This FMMA modification system may provide a tool suitable for studies of intra- and intermolecular interactions of this subunit. Proteins 30:183-192, 1998. © 1998 Wiley-Liss, Inc.
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    Crystal structures and properties of de novo circularly permuted 1,3-1,4-β-glucanases (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 155-167 
    Details
    ISSN: 0887-3585
    Keywords: X-ray diffraction ; protein folding ; genetic engineering ; circular permutation ; 1,3-1,4-β-glucanase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The 1,3-1,4-β-glucanases from Bacillus macerans and Bacillus licheniformis, as well as related hybrid enzymes, are stable proteins comprised of one compact jellyroll domain. Their structures are studied in an effort to reveal the degree of redundancy to which the three-dimensional structure of protein domains is encoded by the amino acid sequence. For the hybrid 1,3-1,4-β-glucanase H(A16-M), it could be shown recently that a circular permutation of the sequence giving rise to the variant cpA16M-59 is compatible with wildtype-like enzymatic activity and tertiary structure (Hahn et al., Proc. Natl. Acad. Sci. USA 91:10417-10421, 1994). Since the circular permutation yielding cpA16M-59 mimicks that found in the homologous enzyme from Fibrobacter succinogenes, the question arose whether de novo circular permutations, not guided by molecular evolution of the 1,3-1,4-β-glucanases, could also produce proteins with native-like fold. The circularly permuted variants cpA16M-84, cpA16M-127, and cpA16M-154 were generated by PCR mutagenesis of the gene encoding H(A16-M), synthesized in Escherichia coli and shown to be active in β-glucan hydrolysis. CpA16M-84 and cpA16M-127 were crystallized in space groups P21 and P1, respectively, and their crystal structures were determined at 1.80 and 2.07 Å resolution. In both proteins the main parts of the β-sheet structure remain unaffected by the circular permutation as is evident from a root-mean-square deviation of main chain atoms from the reference structure within the experimental error. The only major structural perturbation occurs near the novel chain termini in a surface loop of cpA16M-84, which becomes destabilized and rearranged. The results of this study are interpreted to show that: (1) several circular permutations in the compact jellyroll domain of the 1,3-1,4-β-glucanases are tolerated without radical change of enzymatic activity or tertiary structure, (2) the three-dimensional structures of simple domains are encoded by the amino acid sequence with sufficient redundancy to tolerate a change in the sequential order of secondary structure elements along the sequence, and (3) the native N-terminal region is not needed to guide the folding polypeptide chain toward its native conformation. Proteins 30:155-167, 1998. © 1998 Wiley-Liss, Inc.
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    Structural classification of αββ and ββα supersecondary structure units in proteins (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 193-212 
    Details
    ISSN: 0887-3585
    Keywords: secondary structure arrangements ; protein structure database ; left/right topology ; knowledge-based structure prediction ; intrinsic stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α-β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α-β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193-212, 1998. © 1998 Wiley-Liss, Inc.
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    Interaction potentials for protein folding (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 244-248 
    Details
    ISSN: 0887-3585
    Keywords: quasi-chemical ; cost function ; HP model ; Boltzmann statistics ; contact hamiltonian ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We outline a general strategy for determining the effective coarse-grained interactions between the amino acids of a protein from the experimentally derived native-state structures. The method is, in principle, free from any adjustable or empirically determined parameters, and it is tested on simple models and compared with other existing approaches. Proteins 30:244-248, 1998. © 1998 Wiley-Liss, Inc.
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    A model for the nucleotide-binding domains of ABC transporters based on the large domain of aspartate aminotransferase (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 275-286 
    Details
    ISSN: 0887-3585
    Keywords: nucleotide-binding domain ; CFTR ; multidrug resistance ; structure prediction ; P-glycoprotein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: ABC transporters are a large superfamily of integral membrane proteins involved in ATP-dependent transport across biological membranes. Members of this superfamily play roles in a number of phenomena of biomedical interest, including cystic fibrosis (CFTR) and multidrug resistance (P-glycoprotein, MRP). Most ABC transporters are predicted to consist of four domains, two membrane-spanning domains and two cytoplasmic domains. The latter contain conserved nucleotide-binding motifs. Attempts to determine the structure of ABC transporters and of their separate domains are in progress but have not yet been successful.   To aid structure determination and possibly learn more about the domain boundaries, we set out to model nucleotide-binding domains (NBDs) of ABC transporters based on a known structure. Previous attempts to predict the 3D structure of NBDs were based solely on sequence similarity with known nucleotide-binding folds. We have analyzed the sequences of a number of nucleotide-binding domains with the algorithm THREADER, developed by D.T. Jones, and a possible fold was found in the structure of aspartate aminotransferase. We present a model for the N-terminal NBD of CFTR, based on the large domain of the A chain of aspartate aminotransferase. The model is refined using multiple sequence alignment, secondary structure prediction, and 3D-1D profiles. Our model seems to be in good agreement with known properties of nucleotide-binding domains and has some appealing characteristics compared with the previous models. Proteins 30:275-286, 1998. © 1998 Wiley-Liss, Inc.
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    Two structural subdomains of barstar detected by rapid mixing NMR measurement of amide hydrogen exchange (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 295-308 
    Details
    ISSN: 0887-3585
    Keywords: hydrogen exchange mechanism ; denaturants ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Equilibrium amide hydrogen exchange studies of barstar have been carried out at pH 6.7, 32° SDC using one- and two-dimensional nuclear magnetic resonance. An unusually large fraction of the backbone amide hydrogens of barstar exchange too fast to be measured, and the exchange rates of only fifteen slow-exchanging amide sites including indole amides of two tryptophans could be measured in the presence of 0 to 1.8 M guanidine hydrochloride (GdnHCl). Measurement of exchange occurring in tens of seconds in the unfolding transition region was possible by the use of a fast stopped-flow mixing method. The observed exchange rates have been simulated in the EX2 limit according to a two-process model that incorporates two exchange-competent states: a transiently unfolded state (U*) in which many amide hydrogens are completely accessible to solvent-exchange, and a near-native locally unfolded state (N*), in which only one or a few amide hydrogens are completely accessible to solvent-exchange. The two-process model appears to account for the observed exchange behavior over the entire range of GdnHCl concentrations studied. For several measurable slow-exchanging amide hydrogens, the free energies of production of exchange-competent states from the exchange-incompetent native state are significantly higher than the free-energy of production of the equilibrium unfolded state from the native state, when the latter is determined from circular dichroism- or fluorescence-monitored equilibrium unfolding curves. The result implies that U*, which forms transiently in the strongly native-like conditions used for the hydrogen exchange studies, is higher in energy than the equilibrium-unfolded state. The higher energy of this transiently unfolded exchange-competent state can be attributed to either proline isomerization or to the presence of residual structure. On the basis of the free energies of production of exchange-competent states, the measured amide sites of barstar appear to define two structural subdomains - a three-helix unit and a two-β-strand unit in the core of the protein. Proteins 30:295-308, 1998. © 1998 Wiley-Liss, Inc.
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    Characterization of receptors with a new negative image: Use in molecular docking and lead optimization (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 30 (1998), S. 321-336 
    Details
    ISSN: 0887-3585
    Keywords: surface characterization ; DOCK ; structure-based molecular design ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The characterization of receptor binding sites is an important aspect of molecular docking, molecular recognition, and the structure-based design process. This characterization can take several forms: the receptor surface itself can be delineated or described, the space adjacent to the surface can be chemically mapped, or a negative image of the protein binding region can be generated. In this report, we describe a new method of constructing a negative image through generation of a set of spheres. These spheres lie along the receptor surface, and their centers represent possible ligand atom positions. By the method in which they are constructed, these spheres carry a limited amount of energetic and chemical information in addition to their primary geometric information. We test the accuracy of the image by comparing sphere positions to the positions of bound ligand atoms and propose a figure of merit for such tests. Then, we use the spheres to orient ligands in enzyme active sites and show how they can be used to generate low scoring configurations more efficiently than other approaches that search orientation space. In addition, two novel applications of these spheres are described: they are used to help identify structural differences among families of enzymes and to suggest points for ligand modification in analog design. Proteins 30:321-336, 1998. © 1998 Wiley-Liss, Inc.
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    Recognition and interaction of small rings with the ricin A-chain binding site (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 33-41 
    Details
    ISSN: 0887-3585
    Keywords: ricin structure ; inhibitor design ; energy minimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Ricin A-chain is an N-glucosidase that attacks ribosomal RNA at a highly conserved adenine residue. Our recent crystallographic studies show that not only adenine and formycin, but also pterin-based rings can bind in the active site of ricin. For a better understanding of the means by which ricin recognizes adenine rings, the geometries and interaction energies were calculated for a number of complexes between ricin and tautomeric modifications of formycin, adenine, pterin, and guanine. These were studied by molecular mechanics, semi-empirical quantum mechanics, and ab initio quantum mechanical methods. The calculations indicate that the formycin ring binds better than adenine and pterin better than formycin, a result that is consistent with the crystallographic data. A tautomer of pterin that is not in the low energy form in either the gas phase or in aqueous solution has the best interaction with the enzyme. The net interaction energy, defined as the interaction energy calculated in vacuo between the receptor and an inhibitor minus the solvation energy of the inhibitor, provides a good prediction of the ability of the inhibitor to bind to the receptor. The results from experimental and molecular modeling work suggest that the ricin binding site is not flexible and may only recognize a limited range of adenine-like rings. Proteins 31:33-41, 1998. Published 1998 Wiley-Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.
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    Modeling of inhibitor-metalloenzyme interactions and selectivity using molecular mechanics grounded in quantum chemistry (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 42-60 
    Details
    ISSN: 0887-3585
    Keywords: quantum chemistry ; molecular mechanics ; inhibitor ; metalloenzyme complexes ; selectivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We investigated the binding properties of the metalloprotease inhibitors hydroxamate, methanethiolate, and methylphosphoramidate to a model coordination site occurring in several Zn2+ metalloproteases, including thermolysin. This was carried out using both the SIBFA (sum of interactions between fragments ab initio-computed) molecular mechanics and the SCF/MP2 procedures for the purpose of evaluating SIBFA as a metalloenzyme modeling tool. The energy-minimized structures were closely similar to the X-ray crystallographic structures of related thermolysin-inhibitor complexes. We found that selectivity between alternative geometries and between inhibitors usually stemmed from multiple interaction components included in SIBFA. The binding strength sequence is hydroxamate 〉 methanethiolate ≥ methylphosphoramidate from multiple interaction components included in SIBFA. The trends in interaction energy components, rankings, and preferences for mono- or bidentate binding were consistent in both computational procedures. We also compared the Zn2+ vs. Mg2+ selectivities in several other polycoordinated sites having various “hard” and “soft” qualities. This included a hexahydrate, a model representing Mg2+/Ca2+ binding sites, a chlorophyll-like structure, and a zinc finger model. The latter three favor Zn2+ over Mg2+ by a greater degree than the hydrated state, but the selectivity varies widely according to the ligand “softness.” SIBFA was able to match the ab initio binding energies by 〈2%, with the SIBFA terms representing dispersion and charge-transfer contributing the most to Zn2+/Mg2+ selectivity. These results showed this procedure to be a very capable modeling tool for metalloenzyme problems, in this case giving valuable information about details and limitations of “hard” and “soft” selectivity trends. Proteins 31:42-60, 1998. © 1998 Wiley-Liss, Inc.
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    Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 61-73 
    Details
    ISSN: 0887-3585
    Keywords: mutagenesis ; protein stability ; salt bridge ; protein folding ; malic enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A double mutant (R9E/M17K) of pigeon liver malic enzyme with glutamate and lysine replaced for arginine and methionine at positions 9 and 17, respectively, was found to be much more stable in urea and thermal denaturation, but was enzymatically less active than the wild-type enzyme (WT). Unfolding of the enzyme by urea produced a large red shifting of the protein fluorescence maximum from 320 to 360 nm, which was completely reversible upon dilution. Analysis of the denaturation curves monitored by enzyme activity lost suggested that a putative intermediate was involved in the denaturation process. The half unfolding urea concentration, measured by fluorescence spectral changes, increased from 2.24 M for WT to 3.13 M for R9E/M17K. The melting temperature increased by approximately 10°C for R9E/M17K compared with that for WT. Kinetic analysis of the thermal inactivation at 58°C also conformed to a three-state model with the rate constant for the intermediate state of R9E/M17K (k2 = 0.03 min-1) being much smaller than the WT value (k2= 2.39 min-1). Results obtained from single mutants indicated that the decreasing enzyme activity of R9E/M17K was exclusively due to R9 mutation, which increased the KmMn and KmMal by at least one order of magnitude compared with WT. Consequently, a decrease occurred in the specificity constant [kcat/(KmMnKmNADPKmMal)] for the R9 mutants at least four orders of magnitude smaller than the WT. M17K has similar properties to the WT, while R9E is more labile than the WT enzyme. The above results indicate that the extra stability gained by the double mutant possibly occurs through the introduction of an extra ion-pair between E9 and K17, which freezes the double mutant in the putative intermediate state. Examination of the N-terminal amino acid sequence of pigeon liver malic enzyme reveals that position 15 is also a lysine residue. Since the R9E mutant, which has an extra Glu9-Lys15 ion-pair, is less stable than the WT, we conclude that the contribution to malic enzyme stability is specific for the Glu9-Lys17 ion-pair. Proteins 31:61-73, 1998. © 1998 Wiley-Liss, Inc.
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    Prediction of the three-dimensional structure of proteins using the electrostatic screening model and hierarchic condensation (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 74-96 
    Details
    ISSN: 0887-3585
    Keywords: Monte Carlo minimizations in torsion space ; prediction of secondary structure ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We describe a method for predicting the three-dimensional (3-D) structure of proteins from their sequence alone. The method is based on the electrostatic screening model for the stability of the protein main-chain conformation. The free energy of a protein as a function of its conformation is obtained from the potentials of mean force analysis of high-resolution x-ray protein structures. The free energy function is simple and contains only 44 fitted coefficients. The minimization of the free energy is performed by the torsion space Monte Carlo procedure using the concept of hierarchic condensation. The Monte Carlo minimization procedure is applied to predict the secondary, super-secondary, and native 3-D structures of 12 proteins with 28-110 amino acids. The 3-D structures of the majority of local secondary and super-secondary structures are predicted accurately. This result suggests that control in forming the native-like local structure is distributed along the entire protein sequence. The native 3-D structure is predicted correctly for 3 of 12 proteins composed mainly from the α-helices. The method fails to predict the native 3-D structure of proteins with a predominantly β secondary structure. We suggest that the hierarchic condensation is not an appropriate procedure for simulating the folding of proteins made up primarily from β-strands. The method has been proved accurate in predicting the local secondary and super-secondary structures in the blind ab initio 3-D prediction experiment. Proteins 31:74-96, 1998. © 1998 Wiley-Liss, Inc.
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    Erratum: Octanol-Water partition of nonzwitterionic peptides: Predictive power of a molecular size-based model. Proteins 30:86-99, 1998 (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 104-104 
    Details
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Buchwald, P., Bodor, N. Octanol-Water Partition of Nonzwitterionic Peptides: Predictive Power of a Molecular Size-Based Model. Proteins 30:86-99, 1998.Equation 2 should read: P = (Cin - Cfin) Vw/Cfin Vo.In the printed version, the volume ratio (Vw/Vo) incorrectly divides, and not multiplies, the concentration ratio.The publisher apologizes for this error.
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    Prediction and classification of domain structural classes (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 97-103 
    Details
    ISSN: 0887-3585
    Keywords: α domains ; β domains ; α/β domains ; α+β domains ; resubstitution ; jackknife ; SCOP database ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Can the coupling effect among different amino acid components be used to improve the prediction of protein structural classes? The answer is yes according to the study by Chou and Zhang (Crit. Rev. Biochem. Mol. Biol. 30:275-349, 1995), but a completely opposite conclusion was drawn by Eisenhaber et al. when using a different dataset constructed by themselves (Proteins 25:169-179, 1996). To resolve such a perplexing problem, predictions were performed by various approaches for the datasets from an objective database, the SCOP database (Murzin, Brenner, Hubbard, and Chothia. J. Mol. Biol. 247:536-540, 1995). According to SCOP, the classification of structural classes for protein domains is based on the evolutionary relationship and on the principles that govern the 3D structure of proteins, and hence is more natural and reliable. The results from both resubstitution tests and jackknife tests indicate that the overall rates of correct prediction by the algorithm incorporated with the coupling effect among different amino acid components are significantly higher than those by the algorithms without using such an effect. It is elucidated through an analysis that the main reasons for Eisenhaber et al. to have reached an opposite conclusion are the result of (1) misusing the component-coupled algorithm, and (2) using a conceptually incorrect rule to classify protein structural classes. The formulation and analysis presented in this article are conducive to clarify these problems, helping correctly to apply the prediction algorithm and interpret the results. Proteins 31:97-103, 1998. © 1998 Wiley-Liss, Inc.
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    Urea effects on protein stability: Hydrogen bonding and the hydrophobic effect (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 107-115 
    Details
    ISSN: 0887-3585
    Keywords: calorimetry ; desolvation ; linear extrapolation model ; binding ; denaturation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The effects of urea on protein stability have been studied using a model system in which we have determined the energetics of dissolution of a homologous series of cyclic dipeptides into aqueous urea solutions of varying concentration at 25°C using calorimetry. The data support a model in which urea denatures proteins by decreasing the hydrophobic effect and by directly binding to the amide units via hydrogen bonds. The data indicate also that the enthalpy of amide hydrogen bond formation in water is considerably higher than previously estimated. Previous estimates included the contribution of hydrophobic transfer of the α-carbon resulting in an overestimate of the binding between urea and the amide unit of the backbone and an underestimate of the binding enthalpy. Proteins 31:107-115, 1998. © 1998 Wiley-Liss, Inc.
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    Domain motions in bacteriophage T4 lysozyme: A comparison between molecular dynamics and crystallographic data (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 116-127 
    Details
    ISSN: 0887-3585
    Keywords: molecular dynamics ; X-ray crystallography ; essential dynamics ; lysozyme ; hinge bending ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A comparison of a series of extended molecular dynamics (MD) simulations of bacteriophage T4 lysozyme in solvent with X-ray data is presented. Essential dynamics analyses were used to derive collective fluctuations from both the simulated trajectories and a distribution of crystallographic conformations. In both cases the main collective fluctuations describe domain motions. The protein consists of an N- and C-terminal domain connected by a long helix. The analysis of the distribution of crystallographic conformations reveals that the N-terminal helix rotates together with either of these two domains. The main domain fluctuation describes a closure mode of the two domains in which the N-terminal helix rotates concertedly with the C-terminal domain, while the domain fluctuation with second largest amplitude corresponds to a twisting mode of the two domains, with the N-terminal helix rotating concertedly with the N-terminal domain. For the closure mode, the difference in hinge-bending angle between the most open and most closed X-ray structure along this mode is 49 degrees. In the MD simulation that shows the largest fluctuation along this mode, a rotation of 45 degrees was observed. Although the twisting mode has much less freedom than the closure mode in the distribution of crystallographic conformations, experimental results suggest that it might be functionally important. Interestingly, the twisting mode is sampled more extensively in all MD simulations than it is in the distribution of X-ray conformations. Proteins 31:116-127, 1998. © 1998 Wiley-Liss, Inc.
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    Relationships between protein sequence and structure patterns based on residue contactsThis work was done while J.S. was a visiting scientist at EMBL. (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 172-185 
    Details
    ISSN: 0887-3585
    Keywords: sequence-to-structure correlation ; contact environment ; contact prediction ; Bayesian classification ; cluster analysis ; nearest-neighbor classification ; decision tree classification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The identification of correlations between sequence patterns and structural motifs is a prerequisite in the development of protein structure prediction methods. The prediction accuracy indicates whether these correlations are discerned. We present an approach to identify long-range relationships between sequence patterns and structural motifs by varying the granulation of the structure description. Since interaction among residues is a major determinant in protein folding, we consider contact environments formed by two triplets of three sequentially neighboring residues and described by vectors whose components express contact strengths on an atomic level. Through testing various classification schemes, including their resolution and optimizing parameters, discernible relationships between sequences and folds are explored. About ten structural contact states, together with information from noncontacting regions, could improve the accuracy of contact prediction. Proteins 31:172-185, 1998. © 1998 Wiley-Liss, Inc.
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    Comparison of solution and crystal structures of maize nonspecific lipid transfer protein: A model for a potential in vivo lipid carrier protein (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 160-171 
    Details
    ISSN: 0887-3585
    Keywords: lipid binding ; lipid transfer protein ; maize ; molecular modeling ; NMR ; X-ray ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The three-dimensional solution structure of maize nonspecific lipid transfer protein (nsLTP) obtained by nuclear magnetic resonance (NMR) is compared to the X-ray structure. Although both structures are very similar, some local structural differences are observed in the first and the fourth helices and in several side-chain conformations. These discrepancies arise partly from intermolecular contacts in the crystal lattice. The main characteristic of nsLTP structures is the presence of an internal hydrophobic cavity whose volume was found to vary from 237 to 513 Å3 without major variations in the 15 solution structures. Comparison of crystal and NMR structures shows the existence of another small hollow at the periphery of the protein containing a water molecule in the X-ray structure, which could play an important structural role. A model of the complexed form of maize nsLTP by α-lysopalmitoylphosphatidylcholine was built by docking the lipid inside the protein cavity of the NMR structure. The main structural feature is a hydrogen bond found also in the X-ray structure of the complex maize nsLTP/palmitate between the hydroxyl of Tyr81 and the carbonyl of the lipid. Comparison of 12 primary sequences of nsLTPs emphasizes that all residues delineating the cavities calculated on solution and X-ray structures are conserved, which suggests that this large cavity is a common feature of all compared plant nsLTPs. Furthermore several conserved basic residues seem to be involved in the stabilization of the protein architecture. Proteins 31:160-171, 1998. © 1998 Wiley-Liss, Inc.
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    Conformational mapping of a viral fusion peptide in structure-promoting solvents using circular dichroism and electrospray mass spectrometry (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 33 (1998), S. 38-49 
    Details
    ISSN: 0887-3585
    Keywords: MS/MS electrospray mass spectrometry ; CD ; human immunodeficiency virus (HIV) ; glycoprotein 41,000 (gp41) ; N-terminal domain ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The N-terminal domain of human immunodeficiency virus (HIV)-1 glycoprotein 41,000 (FP; residues 1-23; NH2-AVGIGALFLGFLGAAGSTMGARS-CONH2) is involved in the fusion and cytolytic processes underlying viral-cell infection. Here, we use circular dichroism (CD) spectroscopy, along with electrospray ionization (ESI) mass spectrometry and tandem (MS/MS) mass spectrometry during the course of hydrogen/deuterium exchange, to probe the local conformations of this synthetic peptide in two membrane mimics. Since amino acids that participate in defined secondary structure (i.e., α-helix or β-sheet) exchange amido hydrogens more slowly than residues in random structures, deuterium exchange was combined with CD spectroscopy to map conformations to specific residues. For FP suspended in the highly structure-promoting solvent hexafluoroisopropanol (HFIP), CD spectra indicated high α-helix and disordered structures, whereas ESI and MS/MS mass spectrometry indicated that residues 5-15 were α-helical and 16-23 were disordered. For FP suspended in the less structure-promoting solvent trifluoroethanol (TFE), CD spectra showed lower α-helix, with ESI and MS/MS mass spectrometry indicating that only residues 9-15 participated in the α-helix. These results compare favorably with previous two-dimensional nuclear magnetic resonance studies on the same peptide. Proteins Suppl. 2:38-49, 1998. © 1998 Wiley-Liss, Inc.
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    Mass spectrometric mapping of ion channel proteins (porins) and identification of their supramolecular membrane assembly (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 33 (1998), S. 63-73 
    Details
    ISSN: 0887-3585
    Keywords: MALDI mass spectrometric peptide mapping ; membrane proteins ; in situ gel digestion ; porin ; permeability transition ; noncovalent complexes ; protein interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions. Proteins Suppl. 2:63-73, 1998. © 1998 Wiley-Liss, Inc.
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    Fragment-based modeling of NAD binding to the catalytic subunits of diphtheria and pertussis toxins (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 282-298 
    Details
    ISSN: 0887-3585
    Keywords: diphtheria toxin ; docking ; ligand design ; molecular recognition ; NAD ; pertussis toxin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We describe a novel application of a fragment-based ligand docking technique; similar methods are commonly applied to the de novo design of ligands for target protein binding sites. We have used several new flexible docking and superposition tools, as well as a more conventional rigid-body (fragment) docking method, to examine NAD binding to the catalytic subunits of diphtheria (DT) and pertussis (PT) toxins, and to propose a model of the NAD-PT complex. Docking simulations with the rigid NAD fragments adenine and nicotinamide revealed that the low-energy dockings clustered in three distinct sites on the two proteins. Two of the sites were common to both fragments and were related to the structure of NAD bound to DT in an obvious way; however, the adenine subsite of PT was shifted relative to that of DT. We chose adenine/nicotinamide pairs of PT dockings from these clusters and flexibly superimposed NAD onto these pairs. A Monte Carlo-based flexible docking procedure and energy minimization were used to refine the modeled NAD-PT complexes. The modeled complex accounts for the sequence and structural similarities between PT and DT and is consistent with many results that suggest the catalytic importance of certain residues. A possible functional role for the structural difference between the two complexes is discussed. Proteins 31:282-298, 1998. © 1998 Wiley-Liss, Inc.
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    Extracting contact energies from protein structures: A study using a simplified model (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 299-308 
    Details
    ISSN: 0887-3585
    Keywords: quasi-chemical approximation ; statistical potential ; energy landscape ; glass transition ; threading ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In this study, we exploited an elementary 2-dimensional square lattice model of HP polymers to test the premise of extracting contact energies from protein structures. Given a set of prespecified energies for H-H, H-P, and P-P contacts, all possible sequences of various lengths were exhaustively enumerated to find sequences that have unique lowest-energy conformations. The lowest-energy structures (or native structures) of such (native) sequences were used to extract contact energies using the Miyazawa-Jernigan procedure and here-defined reference state. The relative magnitudes of the original energies were restored reasonably well, but the extracted contact energies were independent of the absolute magnitudes of the initial energies. We turned to a more detailed characterization of the energy landscapes of the native sequences in light of a new theoretical framework on protein folding. Foldability of such sequences imposes two limits on the absolute value of the prespecified energies: a lower bound entailed by the minimum requirement for thermodynamic stability and an upper bound associated with the entrapment of the chain to local minima. We found that these two limits confine the prespecified energy values to a rather narrow range which, surprisingly, also contains the extracted energies in all the cases examined. These results indicate that the quasi-chemical approximation can be used to connect quantitatively the occurrence of various residue-residue contacts in an ensemble of native structures with the energies of the contacts. More importantly, they suggest that the extracted contact energies do contain information on structural stability and can be used to estimate actual structural energetics. This study also encourages the use of structure-derived contact energies in threading. The finding that there is a rather narrow range of energies that are optimal for folding a sequence also cautions the use of arbitrary energy Hamiltonion in minimal folding models. Proteins 31:299-308, 1998. © 1998 Wiley-Liss, Inc.
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  • 190
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    Thermal unfolding of small proteins with SH3 domain folding pattern (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 309-319 
    Details
    ISSN: 0887-3585
    Keywords: tyrosine kinase ; protein stability ; differential scanning calorimetry ; CD-spectroscopy ; Sso7d ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 〈 pH 〈 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures.Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. Proteins 31:309-319, 1998. © 1998 Wiley-Liss, Inc.
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  • 191
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    Packing forces in nine crystal forms of cutinase (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 320-333 
    Details
    ISSN: 0887-3585
    Keywords: cutinase ; crystal polymorphism ; packing contacts ; hydrophobicity ; electrostatic and hydrophobic interactions ; protein-accessible surface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: During the characterization of mutants and covalently inhibited complexes of Fusarium solani cutinase, nine different crystal forms have been obtained so far. Protein mutants with a different surface charge distribution form new intermolecular salt bridges or long-range electrostatic interactions that are accompanied by a change in the crystal packing. The whole protein surface is involved in the packing contacts and the hydrophobicities of the protein surfaces in mutual contact turned out to be noncorrelated, which indicates that the packing interactions are nonspecific. In the case of the hydrophobic variants, the packing contacts showed some specificity, as the protein in the crystal tends to form either crystallographic or noncrystallographic dimers, which shield the hydrophobic surface from the solvent. The likelihood of surface atoms to be involved in a crystal contact is the same for both polar and nonpolar atoms. However, when taking areas in the 200-600 Å2 range, instead of individual atoms, the either highly hydrophobic or highly polar surface regions were found to have an increased probability of establishing crystal lattice contacts. The protein surface surrounding the active-site crevice of cutinase constitutes a large hydrophobic area that is involved in packing contacts in all the various crystalline contexts. Proteins 31:320-333, 1998. © 1998 Wiley-Liss, Inc.
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  • 192
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    Theory of kinetic partitioning in protein folding with possible applications to prions (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 335-344 
    Details
    ISSN: 0887-3585
    Keywords: conformational transition ; protein folding ; lattice simulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: This study focuses on the phenomenon of kinetic partitioning when a polypeptide chain has two ground-state conformations, one of which is kinetically more reachable than the other. We designed sequences for lattice model proteins with two different conformations of equal energy corresponding to the global energy minimum. Folding simulations revealed that one of these conformations was indeed much more kinetically accessible than the other. We found that the number and strength of local contacts in the ground-state conformation are the major factors that determine which conformation is reached faster; the greater the number of local contacts, the more kinetically reachable a conformation is. We present simple statistical-mechanical arguments to explain these findings. Our results may be relevant in explaining the phenomenology of such proteins as human plasminogen activator inhibitor-1 (PAI-1), photosystem II, and prions. Proteins 31:335-344, 1998. © 1998 Wiley-Liss, Inc.
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  • 193
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    Structural model of Dex protein from Penicillium minioluteumand its implications in the mechanism of catalysis (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 345-354 
    Details
    ISSN: 0887-3585
    Keywords: DEX gene ; dextranase ; protein threading ; structure prediction ; circular dichroism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The DEX gene encodes an extracellular dextranase (EC 3.2.1.11); this enzyme hydrolyzes the α(1,6) glucosidic bond contained in dextran to release small isomaltosaccharides. Sequence analysis has revealed only one homologous sequence, CB-8 protein, from Arthrobacter sp., with 30% sequence identity. The secondary structure prediction for Dex was corroborated by circular dichroism measurements. To explore the possibility that Dex protein might adopt a fold similar to any known structure, we conducted a threading search of a three-dimensional structure database. This search revealed that the Dex sequence is compatible with the galactose oxidase/methanol dehydrogenase/sialidase fold. A structural model of Dex based on these results is physically and biologically plausible and leads to testable predictions, including the prediction that Asp246 and Glu299 might be catalytic residues. Also, according to this model the Dex enzyme has a mechanism of hydrolysis with net inversion of anomeric configuration. Proteins 31:345-354, 1998. © 1998 Wiley-Liss, Inc.
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  • 194
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    The role played by environmental residues on sidechain torsional angles within homologous families of proteins: A new method of sidechain modeling (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 355-369 
    Details
    ISSN: 0887-3585
    Keywords: homology modeling ; database searching ; conserved torsional angles ; prediction of sidechain conformations ; homologous families of proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We investigated the conservation of sidechain conformation for each residue within a homologous family of proteins in the Protein Data Bank (PDB) and performed sidechain modeling using this information. The information was represented by the probability of conserved sidechain torsional angles obtained from many families of proteins, and these were calculated for a pair of residues at topologically equivalent positions as a result of structural alignment. Probabilities were obtained for a pair of same amino acids and for a pair of different amino acids. The correlation between environmental residues and the fluctuation of probability was examined for the pair of same amino acid residues, and the simple probability was calculated for the pair of different amino acids. From the results on the same amino acid pairs, 17 amino acids, except for Ala, Gly, and Pro, were divided into two types: those that were influenced and those that were not influenced by the environmental residues. From results on different amino acid pairs, a replacement between large residues, such as Trp, Phe, and Tyr, was performed assuming conservation of their torsional angles within a homologous family of proteins. We performed sidechain modeling for 11 known proteins from their native and modeled backbones, respectively. With the native backbones, the percentage of the χ1 angle correct within 30° was found to be 67% and 80% for all and core residues, respectively. With the modeled backbones, the percentage of the correct χ1 angle was found to be 60% and 72% for all and core residues, respectively. To estimate an upper limit on the accuracy for predicting sidechain conformations, we investigated the probability of conserved sidechain torsional angles for highly similar proteins having 〉 90% sequence identity and 〈2.5-Å X-ray resolution. In those proteins, 83% of the sidechain conformations were conserved for the χ1 angle. Proteins 31:355-369, 1998. © 1998 Wiley-Liss, Inc.
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  • 195
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    Structural investigation of C4b-binding protein by molecular modeling: Localization of putative binding sites (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 391-405 
    Details
    ISSN: 0887-3585
    Keywords: complement control protein ; protein modeling ; blood coagulation ; C4b-binding protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one β-chain and seven α-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its α-chains and with protein S through its β-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP α-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the α-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions. Proteins 31:391-405, 1998. © 1998 Wiley-Liss, Inc.
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    Species dependence of enzyme-substrate encounter rates for triose phosphate isomerases (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 406-416 
    Details
    ISSN: 0887-3585
    Keywords: electrostatics ; Brownian dynamics ; triose phosphate isomerase ; diffusion-control ; similarity index ; rate enhancement ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Triose phosphate isomerase (TIM) is a diffusion-controlled enzyme whose rate is limited by the diffusional encounter of the negatively charged substrate glyceraldehyde 3-phosphate (GAP) with the homodimeric enzyme's active sites. Translational and orientational steering of GAP toward the active sites by the electrostatic field of chicken muscle TIM has been observed in previous Brownian dynamics (BD) simulations. Here we report simulations of the association of GAP with TIMs from four species with net charges at pH 7 varying from -12e to +12e. Computed second-order rate constants are in good agreement with experimental data. The BD simulations and computation of average Boltzmann factors of substrate-protein interaction energies show that the protein electrostatic potential enhances the rates for all the enzymes. There is much less variation in the computed rates than might be expected on the basis of the net charges. Comparison of the electrostatic potentials by means of similarity indices shows that this is due to conservation of the local electrostatic potentials around the active sites which are the primary determinants of electrostatic steering of the substrate. Proteins 31:406-416, 1998. © 1998 Wiley-Liss, Inc.
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  • 197
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    Functional conformational changes of endo-1,4-xylanase II from Trichoderma reesei: A molecular dynamics study (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 434-444 
    Details
    ISSN: 0887-3585
    Keywords: hinge ; structural change ; xylose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recent crystallographic studies have revealed a range of structural changes in the three-dimensional structure of endo-1,4-xylanase (XYNII) from Trichoderma reesei. The observed conformational changes can be described as snapshots of an open-close movement of the active site of XYNII. These structures were further analyzed in this study. In addition, a total of four 1 ns molecular dynamics (MD) simulations were performed representing different states of the enzyme. A comparison of the global and local changes found in the X-ray structures and the MD runs suggested that the simulations reproduced a similar kind of active site opening and closing as predicted by the crystal structures. The open-close movement was characterized by the use of distance difference matrixes and the Hingefind program (Wriggers and Schulten, Proteins 29:1-14, 1997) to be a ‘hinge-bending’ motion involving two large rigidly-moving regions and an extended hinge. This conformational feature is probably inherent to this molecular architecture and probably plays a role in the function of XYNII. Proteins 31:434-444, 1998. © 1998 Wiley-Liss, Inc.
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  • 198
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    Interaction of human SRY protein with DNA: A molecular dynamics study (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 417-433 
    Details
    ISSN: 0887-3585
    Keywords: molecular dynamics ; sex-determining region Y (SRY) protein ; high mobility group (HMG) box ; DNA-binding proteins ; DNA bending ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecular dynamics simulations have been conducted to study the interaction of human sex-determining region Y (hSRY) protein with DNA. For this purpose, simulations of the hSRY high mobility group (HMG) domain (hSRY-HMG) with and without its DNA target site, a DNA octamer, and the DNA octamer alone have been carried out, employing the NMR solution structure of hSRY-HMG-DNA complex as a starting model. Analyses of the simulation results demonstrated that the interaction between hSRY and DNA was hydrophobic, just a few hydrogen bonds and only one water molecule as hydrogen-bonding bridge were observed at the protein-DNA interface. These two hydrophobic cores in the hSRY-HMG domain were the physical basis of hSRY-HMG-DNA specific interaction. They not only maintained the stability of the complex, but also primarily caused the DNA deformation. The salt bridges formed between the positive-charged residues of hSRY and phosphate groups of DNA made the phosphate electroneutral, which was advantageous for the deformation of DNA and the formation of a stable complex. We predicted the structure of hSRY-HMG domain in the free state and found that both hSRY and DNA changed their conformations to achieve greater complementarity of geometries and properties during the binding process; that is, the protein increased the angle between its long and short arms to accommodate the DNA, and the DNA became bent severely to adapt to the protein, although the conformational change of DNA was more severe than that of the hSRY-HMG domain. The sequence specificity and the role of residue Met9 are also discussed. Proteins 31:417-433, 1998. © 1998 Wiley-Liss, Inc.
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  • 199
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    Density functional studies on herpes simplex virus type 1 thymidine kinase-substrate interactions: The role of Tyr-172 and Met-128 in thymine fixation (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 453-459 
    Details
    ISSN: 0887-3585
    Keywords: quantum mechanical calculations ; substrate-enzyme interactions ; mutants ; functional role of amino acids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The enzyme herpes simplex virus type 1 thymidine kinase (HSV1 TK) salvages thymidine into the DNA metabolism of the virus. In the active site, the thymine ring of the nucleoside binds in a pocket, formed by two residues, Tyr-172 and Met-128, in a sandwich-type orientation. To investigate the nature of the thymine-enzyme pocket interactions, we have carried out density functional theory calculations with gradient-corrected exchange-correlation functionals of models of the thymine-HSV1 TK adduct. Our calculations indicate that the role of Met-128 in the substrate fixation is purely steric and hydrophobic, while the substrate-Tyr-172 interaction is essentially electrostatic in nature. These findings are completely consistent with the available catalytic properties of mutants on the 128 position. Proteins 31:453-459, 1998. © 1998 Wiley-Liss, Inc.
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    Electrostatics, allostery, and activity of the yeast chorismate mutase (1998)
    New York, NY : Wiley-Blackwell
    Proteins: Structure, Function, and Genetics 31 (1998), S. 445-452 
    Details
    ISSN: 0887-3585
    Keywords: chorismate mutase ; activity ; allosteric ; electrostatics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The predicted active site of chorismate mutase of baker's yeast Saccharomyces cerevisiae has been studied by continuum electrostatics, molecular surface/volume calculations, and molecular modeling. Our study shows that despite being subject to an allosteric transition, the enzyme's active-site pocket neither decreased in volume nor deformed significantly in shape between the active R state and the inactive T state. We find that the polar atmosphere in the pocket is responsible for the enzyme's affinity. A single amino acid, Glu23, can adequately account for the atmospheric variation. This residue swings into the active-site pocket from the R state to the T state. In the R state, Glu23 on helix H2 doubly pairs with Arg204 and Lys208 of H11, which is packed against H2. In the T state, a slide occurs between H11 and H2 such that Glu23 can no longer interact with Lys208 and competes with Asp24 for interacting with Arg204. Consequently, Glu23 is found in the T state to couple with Arg157, an active-site residue critical to substrate binding. The tandem sliding of H11 in both monomers profoundly changes the interactions in the dimer interface. The loop between H11 and H12 demonstrates the largest conformational change. Hence, we establish a connection between the allosteric transition and the activity of the enzyme. The conformational change in the transition is suggested to propagate into the active-site pocket via a series of polar interactions that result in polarity reversal in the active-site pocket, which regulates the enzyme's activity. Proteins 31:445-452, 1998. © 1998 Wiley-Liss, Inc.
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