Bibliothek

Notizen: Conformational energy computations were carried out on collagenlike triple-stranded conformations of several poly(tripeptide)s with the general structure CH3CO—(Gly—X—Y)3—NHCH3. The sequences considered had various amino acid residues in position X or Y of the central tripeptide, with either Pro or Ala as a neighbor, i.e., Gly-X-Pro, Gly-X-Ala, Gly-Pro-Y, and Gly-Ala-Y. Minimum-energy conformations were computed for the side chains, and their distributions were compared for the four sequences. The residues used were Abu (= α-aminobutyric acid), Leu, Phe, Ser, Asp, Asn, Val, Ile, and Thr. The conformational energy of a —Ch2—CH3 side chain in Abu was mapped as a function of the dihedral angle χ1. Intrastrand interactions with neighboring residues do not affect the conformations of a side chain in position Y, and they have a minor effect on it in the X-Ala sequence, but they strongly restrict the conformational freedom of the side chain in the X-Pro sequence. Conversely, interstrand interactions do not affect side chains in position X, but they strongly restrict the conformational freedom of a side chain in position Y if there is a nearby Pro residue in a neighboring strand. Hydrogen bonds with the backbone can be formed in some conformations of long polar side chains, such as Asp, Asn, or Gln. All amino acid residues can be accommodated in collagen. Because of the interactions mentioned above, steric and energetic constraints can be correlated with observed preferences of certain amino acids for positions X or Y in collagen. Hence, these preferences may be explained, in part, in terms of differences in the conformational freedom of the side chains in the triple-stranded structure.

Notizen: The ionization constants of the tyrosyl groups of chymotrypsinogen and of nitrated-chymotrypsinogen (two tyrosyl residues nitrated) have been determined by difference spectrophotometry. In chymotrypsinogen, two of the four tyrosyl groups ionize without any time dependence. Above pH greater than ca. 12.5, time-dependent spectral changes are seen for 0.7 group equivalent. The data can be fitted to the values of pK′1 9.75 ± 0.07, pK′2 11.55 ± 0.05, pK′3 13.30 ± 0.05. In nitrated-chymotrypsinogen, the two nitrated tyrosyl residues have pK′1 6.44 and pK′2 8.30. For both proteins, these pK′ values are in agreement with those evaluated from potentiometric titration and calorimetric data using computer-assisted curve-fitting analysis.

Notizen: The conformation of several samples of poly(α,β-L-Asp) with a molar fraction of β-bonds ranging from 0.1 to 0.55 was investigated by means of ir and CD spectroscopy and potentiometric titration and compared with the results obtained previously with poly(α-L-Asp). All samples investigated underwent a conformational change induced by changes in their degree of ionization: unpronounced ir absorption of amide V at 650 cm-1 was shifted to 620 cm-1 and substantially increased on deionization; CD spectra changed with the degree of ionization, passing through an isosbestic point; and the pattern of the titration curves was more complex than that of a simple polyelectrolyte. The conformation developing with the decreasing degree of ionization may be considered to be α-helix, as deduced according to the analogous behavior of other polypeptides. The extent of the conformational change in the individual samples depends on the molar fraction of β-bonds: the higher it is, the lower is the helix-forming ability of the sample.

Notizen: The use of 1H-nmr spectroscopy is demonstrated to be a useful analytical method to characterize the structure of synthetic peptides attached to soluble, macromolecular polyoxyethylene (POE) supports in the liquid-phase method (LPM) of peptide synthesis. We report an extensive 360-MHz 1H-nmr study of POE-bound homo-oligo-L-methionine peptides. A combination of high field and selective saturation or Redfield pulse methods allows resolution of individual backbone NH and α-CH resonances of dilute peptides in the presence of strong resonances from macromolecular POE and/or protonated solvents. The nmr spectra for the POE-bound peptides in CDCl3 are qualitatively similar to those of the low-molecular-weight Boc-L-Metn-OMe peptide esters. This corroborates other observations that POE has little effect on peptide stucture. The backbone α-CH region of peptides is overlapped by signals from the terminal oxyethylene group of POE, but the peptide side-chain and low-field backbone NH resonances are well resolved. In trifluoroethanol the Boc-(L-Met)n-NH-POE heptamer and octamer adopt the right-handed α-helical structure, and the present nmr studies provide evidence for two strong intramolecular hydrogen bonds to stabilize the helices. In water, the N-deblocked derivatives, (L-Met)n-NH-POE oligomers adopt β-sheet structure and manifest well-resolved nonequivalent NH resonances with 6-7 Hz 3JNH-CH coupling constants.

Notizen: The concerted model of Monod, Wyman, and Changeux is generalized so that all effects of interactions for an enzyme operating at a nonequilibrium stationary state are considered. In contrast to the original model, which is based on an analogy to equilibrium ligand binding, the generalization may show both “positive” and “negative cooperativity” in both catalytic binding and conformational processes. Furthermore, in contrast to any equilibrium binding model, the Hill coefficients may be greater than the number of sites n. For catalysis, the maximum value is 2n, and for conformational changes, n + 1. These points are illustrated by two cases that yield simpler analytic expressions. The first obtains when catalysis occurs on a much faster time scale than the conformational changes, and the second, when this situation is reversed.

Notizen: By combining gel permeation chromatography (GPC) and light-scattering spectroscopy, including photon correlation and angular distribution of absolute scattered intensity, we were able to characterize immunologically active Haemophilus influenzae type b polysaccharide (HIB Ps) bovine serum albumin (BSA) conjugates in terms of equivalent hydrodynamic radius rh ∼ (6.2 ± 0.6) × 102 Å, apparent radius of gyration rg ∼ (5.4 ± 0.3) × 102 Å, apparent molecular weight Mw ∼ (3.5 ± 0.4) × 106 g/mol, and a second virial coefficient A2 ∼ (1.9 ± 0.3) × 10-4 cm3 mol/g2. We could study the effects of each of the processes in the conjugate formation according to the following procedure: BSA (dialysis, modification, fractionation) + HIB Ps → HIB Ps/BSA conjugate (conjugate formation, fractionation). Narrow distributions of HIB Ps BSA conjugate formation can be achieved using fractionated BSA.

Notizen: We have measured the ir absorption of 5′CMP, 5′IMP, and poly(I)·poly(C) from ∼25 to ∼500 cm-1. From a comparison of the data with the previously measured absorption of the corresponding nucleosides and bases we can identify several “lines” associated with the deformation of the ribose ring. Out-of-plane deformation of the bases contributes strongly to vibrations near 200 cm-1. The same ribose vibrations observed in the nucleotides are found in poly(I)·poly(C). They sharpen with increasing water absorption. A study of the spectra of poly(I)·poly(C) as a function of the adsorbed water indicates that water does not contribute in a purely additive fashion to the polynucleotide spectrum but depends on the conformation of the helix. However, the only spectral feature that shifts drastically with conformation is near 45 cm-1. Measurements at cryogenic temperatures indicate some sharpening of the spectrum of poly(I)·poly(C). Instead, no sharpening is observed in the spectrum of the nucleotides. Shear degradation of poly(I)·poly(C) produces significant spectral changes in the 200-cm-1 region and sharpening of the features assigned to the low-frequency ribose-ring vibrations.

Notizen: A study of the oxygen replacement reaction of carbon monoxide-saturated hemoglobin (HbA0) was carried out using spectroscopic, calorimetric, and pH titration methods. Under fully saturated conditions the replacement reaction can be defined by a single partition constant over all ratios of bound oxygen to carbon monoxide. This indicates that under saturating conditions Haldane's first law for the ligand binding of gas mixtures holds for any CO/O2 ratio. It further shows that there is no appreciable difference in relative CO-O2 affinity between the α- and β-chains. The same partition coefficient was found to hold for different pH, buffer, and allosteric effector conditions. The lack of any pH dependence of the partition coefficient was confirmed by the absence of proton changes for the replacement reaction. The temperature dependence of the partition coefficient and calorimetric results yield a value for the enthalpy of the reaction of -3.65 ± 0.29 kcal/mol/heme.

Notizen: We demonstrate that the isotropic absorption and linear dichroism in an unknown flow field can be used to determine base tilt in polynucleotides if three transitions are measured and the directions of the corresponding dipoles are known. The method is applied here to reach conclusions about the base tilt in poly(rA), poly(rA)+·poly(rA), and poly(rC). The respective values are: 28° tilt about the axis + 50° toward C8 from the C1′ → N9, and 25° tilt about the axis + 118° toward C5 from C1′ → N1. The results for poly(rA)+·poly(rA) are consistent with the accepted model. Spectra were measured for poly(rC)+·poly(rC), but definite conclusions must await reliable directions for transition dipoles. The dipole direction for the 218-nm transition in rC is found to be +13° or +43° toward C5 from C1′ → N1. The CD spectra to about 168 nm are presented and discussed.

Notizen: Measurements of stress relaxation in uniaxial extension and associated time-dependent birefringence have been made on bovine fibrin film, prepared by gentle compaction of coarse fibrin clots, containing 13-22% fibrin plasticized with either aqueous buffer or glycerol. Both unligated and ligated (i.e., with α-α and γ-γ ligation by fibrinoligase, factor XIIIa) films were studied. Both types showed two stages of stress relaxation, with time scales of approximately 10 and 103-104 s, respectively, with a plateau region between. In the plateau, the nominal (engineering) stress for ligated glycerol-plasticized film is proportional to In λ, where λ is the stretch ratio, up to λ ≅ 2, and it decreases with increasing temperature. For unligated glycerol-plasticized film, the stresses are smaller by a factor of one-half to one-third. For ligated film, the second stage of relaxation is relatively slight, and recovery after release of stress is often nearly complete. For unligated film, the second stage involves a substantial drop in stress, and after recovery there is a significant permanent set. A second relaxation for ligated film reproduces the first, but for unligated film it reproduces the first only if the initial relaxation is terminated before the second stage; otherwise, the second relaxation shows a weaker structure. The behavior of water-plasticized film is similar to that of glycerol-plasticized except that the second stage of relaxation occurs at shorter times. During the first stage of stress relaxation, up to about 100 s, the birefringence and the stress-optical coefficient increase; during the plateau zone of stress relaxation, the birefringence of ligated films is approximately constant and is proportional to 2λ2/(λ2 + 1) - 1, where λ is the stretch ratio. This dependence is predicted by a two-dimensional model in which rodlike elements in the plane of the film are oriented with independent alignment. During the final stage of stress relaxation, the birefringence of ligated films decreases slightly; that of unligated films decreases substantially, but less rapidly than the stress, corresponding to a further increase in the stress-optical coefficient. With additional information from small-angle x-ray scattering reported in an accompanying paper, the first stage of relaxation is attributed to partial release of bending forces in the fibers by orientation, accompanied by increased birefringence. The second stage is attributed, for ligated films, to an internal transition in the fibrin units accompanied by elongation of some of the fibers; and in the unligated films, to a combination of the latter transition with slippage of protofibrils lengthwise within the fiber bundles that causes some loss of orientation, which diminishes the birefringence.

Notizen: A microscope capable of measuring the CD of intact single eukaryotic cells, DNA microcrystals, and other microscopic structures has been constructed and tested. It can measure the CD spectra in the 200- and 800-nm wavelength range and consists of a modification to a standard Cary 60 CD machine in combination with a Zeiss uv microspectrometer. Preliminary CD spectra of red blood cells and lymphocytes are presented.

Notizen: The distribution of fibers in agarose gels has been studied by electron-microscopic examination of replicas formed from freeze-fracture surfaces. For gels set in water, the results obtained support the model proposed for the gel structure by Arnott et al. (1974) of a random array of long, straight, connected fibers, with each fiber having a diameter equivalent to that of an aggregate of approximately 10-30 agarose helixes, depending on the initial agarose concentration. The density of these fibers, their water content, and the total length of fibers per unit volume have been derived from the measured distribution of intersections per unit area of freeze-fracture surfaces. For gels set in the presence of salt, the distribution of fibers becomes distinctly non-Poissonian, leading to larger interfiber spaces and a gel of greater effective pore size. The larger pore size of gels set in the presence of salt also has been revealed by electrophoretic measurements in which the relative migration rates of plasmid DNA molecules of varying conformations have been determined.

Notizen: Energy embedding has been shown recently to be a useful extension of the distance geometry approach to conformational calculations in the case of very small molecules and simple energy functions. This paper tests the ability of energy embedding to locate low energy conformations satisfying both weak and strong geometric constraints when the molecule is the small protein, bovine pancreatic trypsin inhibitor, and the energy function is the complicated Oobatake-Crippen residue-residue potential. Using the potential function alone, the algorithm reaches a structure with energy lower than that of the native conformation, but with little resemblance to it. Aided by numerous geometric constraints, such as preformed secondary structure segments, the algorithm again finds a local minimum with energy better than that of the native, and with only 3.3 Å rms deviation from it. This is significantly closer to the native value than can be obtained using standard distance geometry and the geometric constraints alone. Thus, energy embedding using the Oobatake-Crippen potential function is a significant help in finding native conformations of proteins. However, additional trials on a hairpin bend fragment of trypsin inhibitor demonstrate the potential's shortcomings in encouraging proper secondary structure.

Notizen: Molecular-mechanics calculations have been carried out on the base-paired deoxy dodecanucleoside undecaphosphates d(CGCGAATTCGCG)2 and d(A12)·d(T12). These refinements were carried out using the model-built Arnott B-DNA geometry as initial coordinates (with a helix repaeat of 10.0 residues/turn), as well as helix repeats ranging from 9 to 12 residues/turn. There was some variation in the optimum calculated helix repeat, depending on the dielectric model, the presence or absence of counterions, and the method used for inclusion for nonbonded interactions; the most interesting general result of these calculations was the coupling between furanose sugar puckering and twist. This coupling was observed for all models. With a helix repeat of 9.0 residues/turn, all sugars remain C(2′)endo after refinement; as the helix repear increases to 12.0 residues/turn, the number of sugars repuckering to O(1′)endo and C(3′)endo increases also. With our most rigorous model (i.e., a model with no cutoff distance for nonbonded interactions) and a helix repeat of 10.0 residues/turn, we find a greater tendency for pyrimidine than purine repuckering in d(CGCGAATTCGCG)2, in agreement with the x-ray structural data of Drew et al. [(1981) Proc. Natl. Acad. Sci. USA 78, 2179-2185].We also carried out a number of calculations in which we “forced” one of two deoxy sugars to repucker or one of the C3′-O3′-P-O5′ (ω) torsion angles to change from gauche- to trans using dihedral angle constraints. After the constraints were removed, some of these structures “reverted” to the sugar pucker of the initial structures, while others remained repuckered. In all cases, the energies for repuckered structures after refinement were very similar to energies of the initial structure. Experiments and theory suggest that local conformational fluctuations play an essential role in nmr relaxation of 31P and 13C atoms in double-helical DNA. The results of our previous calculations on hexanucleoside phosphates and the calculations presented there are consistent with an important contribution to nmr relaxation processes of conformational changes in the torsion angle ω′ from gauche- to trans and deoxy sugar repuckering from C(2′)endo to C(3′)endo. Specifically, the calculations presented here indicate a very flexible phosphate backbone in helixes having an intermediate helix repeat of 10 to 11 residues/turn. These helixes may accommodate sugars of variable pucker without significantly disrupting base-base hydrogen-bonding and stacking interactions. All of the variant structures are similar in energy, suggesting that conversion between them can occur on a nanosecond time scale, as observed in nmr relaxation experiments.

Notizen: The microwave absorption of aqueous solutions of DNA extracted from E. coli has been studied between 8 and 12 GHz by the use of an optical heterodyne technique. By measuring optically the temperature rise produced in an absorbing sample by pulsed microwave radiation, unambiguous, direct measurement of the microwave absorption is possible. Our results show that E. coli DNA absorbs microwaves in the 8-12-GHz region substantially more efficiently than water, which is itself an extremely efficient absorber. The observed absorption is featureless and decreases slightly with increasing frequency. These observations are consistent with an explanation involving direct absorption by longitudinal acoustic modes of the double helix.

Notizen: The formation of polyelectrolyte complexes of glycol chitosan (GlChi) with sodium salts of dextran sulfate (DS), carboxymethyl cellulose (CMC), polygalacturonic acid (GalUA)n, and alginic acid (AlgA) has been studied. The number of ionizable groups per pyranose ring and the degree of dissociation as a function of pH in the polycation and polyanions have been determined using conductometric and potentiometric titrations. The formation of the complexes at different pH values has been followed by turbidity measurements. It is found that stoichiometric complexes of the polycation with CMC, (GalUA)n, and AlgA are formed at a mixing ratio of 0.5, indicating that the conformation may correspond to chains of equal lengths. In the case of the complex of GlChi with DS, the stoichiometric composition corresponds to a mixing ratio of 0.62. Thin transparent films of the complexes have been obtained by dehydration under reduced pressure on a layer of mercury. The dielectric constant ε′ and loss ε″ of thin films of these complexes have been measured in the range of frequencies of 1-100 kHz at different temperatures above and below room temperature. The GlChi-DS complex shows very little change in the values of ε′ and ε″ with frequency or temperature. On the other hand, films of GlChi-CMC and GlChi-(GalUA)n complexes show a significant increase in ε′ and ε″ as the temperature is increased above room temperature. The increase is more prominent at low frequencies. This behavior is attributed to the Maxwell-Wagner interfacial polarization. In the case of GlChi-AlgA film, the values of ε′ and ε″ increase enormously as the temperature increases. This behavior is similar to the increase in ε′ and ε″ observed by Michels et al. [(1965) J. Phys. Chem. 69, 1456-1465] as the salt concentration increases in another polyelectrolyte complex studied by them. The electric double-layer mechanism proposed by Schwarz [(1962) J. Phys. Chem. 66, 2636-2642] to account for the dielectric properties of biocolloids is shown to account quantitatively for the observations on the GlChi-AlgA film.

Notizen: A melting experiment was performed on the whole set of populations of the replicative form of φX174 DNA, which can be obtained treating this DNA with rat liver nicking-closing enzyme in the presence of ethidium bromide. Gel electrophoresis performed by loading the DNA samples at neutral and alkaline pH allows separation of these populations in discrete sets of bands, which can then be compared. The outcome of the experiments indicates that in the range of electrophoretic mobilities which can be explored, no band is formed exclusively by circular complementary strands which can be separated by alkaline denaturation. These results are compared with what would be expected if double-stranded closed circular DNA had structures other than the canonical double helix. Under nonrestrictive hypotheses, the experiments reported allow one to obtain a minimum estimate of the absolute value of the linking number of a closed circular double-stranded DNA: for native φX174 RF DNA, the linking number appears to be greater than 12 (in absolute value). Some data on the electrophoretic mobility of denatured closed circular duplexes are reported, which still wait for a physicochemical interpretation.

Notizen: Synthetic cyclic octapeptides of general structure cyclo[Glu(γOBzl)-Sar-Gly-(N-R)Gly]2 (R = n-hexyl and cyclohexyl) transport calcium ions selectively across organic phases and phospholipid membranes. We have now used proton nmr spectroscopy (360 MHz) to study the solution conformation(s) of their calcium complexes. When Ca(ClO4)2 was added to solutions of these peptides in CDCl3, nmr spectra of the resulting calcium complexes were characteristic of a single C2-symmetric conformer. From a Karplus-Bystrov analysis of vicinal coupling constants in both the peptide backbone and Glu side chain (treated as an ABCC′MX spin system), in conjuction with model-building studies, a structure was proposed in which the calcium ion is bound in an octahedral-type complex by the four (coplanar) carbonyl groups of the (all-trans) Glu-Sar and Gly-(N-R)Gly peptide bonds. Occurrence of preferred rotamers about Glu side chain Cα-Cβ bonds indicated that restricted rotation in peptide side chains arises upon calcium binding.

Notizen: (L-Cys)n + N-base systems and (L-Cys)n + (L-Lys)n systems were studied by ir spectroscopy. It is shown that in the water-free systems, SH⃛N ⇌ S-⃛H+N hydrogen bonds are formed. With the (L-Cys)n + N-base systems, both proton-limiting structures in the SH⃛N ⇌ S-⃛H+N bonds have equal weight when the pKa of the protonated N-base is 2 pKa units larger than that of (L-Cys)n. The same is true with the water-free (L-Cys)n + (L-Lys)n system. Thus, with regard to the type of proton potentials present, these hydrogen bonds are proton-transfer hydrogen bonds showing very large proton polarizabilities. This is confirmed by the occurrence of continua in the ir spectra. Small amounts of water open these hydrogen bonds and increase the transfer of the proton to (L-Lys)n. In the (L-Lys)n + N-base systems, with increasing proton transfer the backbone of (L-Cys)n changes from antiparallel β-structure to coil. In (L-Cys)n + (L-Lys)n, the conformation is determined by the (L-Lys)n conformation and changes depending on the chain length of (L-Lys)n. Finally, the reactivity increase in the active center of fatty acid synthetase, which should be caused by the shift of a proton, is discussed on the basis of the great proton polarizability of the cysteine-lysine hydrogen bonds.

Notizen: We report electric-dichroism and electron-microscopic studies of chromatin fibers fixed by protein-protein crosslinking at salt concentrations ranging from 10 to 100 mM. The results confirm a progressive disorganization of the fiber as the salt concentration is lowered. The positive dichroism and large polarizability anisotropy characteristic of the 300-Å diameter fiber found in 100 mM salt are replaced by negative dichroism and smaller effective polarizability anisotropy or dipole moment for samples fixed at lower salt concentration. We interpret the results in terms of segmental, field-induced orientation of the disorganized structure which is present in low salt concentrations. We also observed a field-induced absorbance decrease in chromatin fibers fixed at salt concentration at and below 100 mM. All three optical effects, namely overall orientation of the high-salt fixed fiber, segmental orientation of the low-salt fixed fiber, and field-induced absorbance decrease, occur on roughly the same time scale, 20-100 μs for 50 nucleosome polynucleosomes. The polarizability anisotropy of fibers fixed in 100 mM salt was found to be proportional to the length of the fragment and to the reciprocal square root of the conductivity of the solution used for electric-dichroism measurements. Addition of Mg2+ to the measurement buffer affected the dichroism amplitude of samples fixed below 100 mM salt but not those fixed at 100 mM salt. The results reinforce the need for caution in interpreting electric-dichroism measurements on chromatin fibers because of possible field-induced distortion effects.

Notizen: DNA with Mn2+ as the only counterion has been prepared, and the extent of the Mn2+ binding was determined under a variety of conditions through measurements of the proton relaxation enhancement of water. The total extent of Mn2+ binding per DNA phosphate is found to be 0.43 ± 0.04, independent of the metal ion concentration in the experimental range of 2.8 × 10-5 to 2.1 × 10-3M. The predictions of Manning's condensation theory and those obtained from solution of the generalized Poisson-Boltzmann equation regarding the extent of divalent ion binding to polyelectrolytes, in the presence and absence of monovalent counterions, are compared with one another and with the experimental results. Good agreement between the two theoretical approaches is found, with less than 14% variance in the predicted extent of binding over a large range of mono- and divalent ion concentrations. While the predictions of both theoretical approaches generally agree with the experimental results, some discrepancies are noted and their possible origins discussed.

Notizen: The basic formulas for the incorporation into the diffusion-collision model of the stabilities of intermediate states on the folding pathway are derived and discussed. A hypothetical two-step folding pathway is calculated in detail. A model for the production of incorrectly folded intermediates is suggested and some numerical estimates made. Implications and future directions in the evolution of the model are discussed. Three appendices deal with some mathematical aspects of the model.

Notizen: Quasielastic light scattering and electrophoretic light scattering experiments were performed on chicken erythrocyte polynucleosome solutions at various temperatures and ionic strengths. The apparent diffusion coefficient, Dapp, was found to depend on the scattering vector K. In general, Dapp can be described as a damped oscillatory function of K in the ionic strength range of 10 to 60 mM and over the temperature range of 10 to 40°C. Electrophoretic light scattering studies on total digest chromatin samples indicate the apparent charge on the polynucleosomes increases as the ionic strength is lowered from 10 to 1 mM. These data are interpreted in terms of fluctuations in the surface charge distribution of the polyion and subsequent inducement of an asymmetric distribution of small ions about the polyion. These fluctuation components lead to the formation of “clusters” of polyions.

Notizen: Absorption of radiation by DNA polymer is calculated for the case of bent polymer chains. The molecule is assumed to be straight except for localized bends. The region between two bends is studied in particular. The vibrational properties of the bends are parameterized by a transmission and a reflection coefficient. A general Green function expression for absorption is studied for various values of the damping rate, as well as the transmission/reflection coefficients. Curves of absorption vs frequency are shown for a number of cases.

Notizen: When adsorbed from an aqueous dilute solution at high pH into the pores of an inert cellulose acetate filter, poly(α,L-glutamic acid) remains strongly anchored to the pore walls. The existence of the helix-coil transition for the adsorbed polypeptide in a certain pH range is evidenced by static and dynamic membrane properties displayed by the “activated” filter, such as excess cation uptake, membrane potential, and hyraulic permeability. In particular, the variations of the hydrodynamic thickeness present a sigmoidal shape characteristic of the helix-coil transition at the interface, a transition apparently less sharp than in solution.

Notizen: We have studied the linear dichroism (LD) of rat liver chromatin oriented by flow. Soluble chromatin, prepared by brief nuclease digestion, is found to exhibit a positive LD at low ionic strength (1 mM NaCl), with a constant LD/A over the absorption band centered at 260 nm (A, isotropic absorbance). Several previous dichroism studies on soluble chromatin have been performed on sonicated materials and have given negative LD, probably due to the presence of uncoiled DNA. The positive dichroism can be interpreted in terms of a supercoil of DNA in chromatin with a pitch angle larger than 55°, and is, for example, consistent with a model where the cylindrical nucleosome core particles are stacked face to face in the chromatin filament. In contrast to the nuclease-digested chromatin, sonicated chromatin was confirmed to exhibit negative LD. This difference can be attributed to a partial uncoiling of the linker regions between the nucleosomes due to the shearing. The structural transition of chromatin to a compact form can be observed as a reduction of the positive LD of the nuclease-digested chromatin to almost zero in 0.1 M NaCl or in 0.1 mM MgCl2. This transition is due to a decreased electrostatic repulsion between negative phosphate groups on the DNA chain. In the case of Na+, this can be explained as a screening effect due to the bulk concentration of Na+. With Mg2+ a considerably stronger effect may indicate a more localized binding to the phosphates. At ionic strengths higher than 0.5M NaCl, the dissociation of the histones from DNA leads to uncoiling of chromatin. The change in LD during this process shows that histone H1 contributes only to a small degree to the coiling of the DNA chain, whereas histones H3 and H4 play the major role in the coiling.

Notizen: On the basis of measurements of enthalpy of dissociation and of dilution, an interamolecular conformational transition induced by pH change is shown for pectic acid in aqueous solution. Additional evidence is given by potentiometic, viscometric, and chiroptical results. The transition from a more rigid, probably H-bonded, structure prevailing at low pH to a more extended one at around neutrality is accompanied by a ΔH value of about 500 cal/equiv and a ΔS value of 1.6 cal/equiv K in water at 25°C. The addition of salts increases the stability of the rigid conformation without changing the general features of the phenomenon. Dilatometric measurements suggest that the transition is accompanied by practically no change in the overall solvation of the polymer chain.

Notizen: Amphotericin B, a polyene macrolide antibiotic, exists in aqueous solution as a poorly soluble, high-molecular-weight aggregate. A borate complex of this polyene was prepared that has greater solubility and is less aggregated. In aqueous solution this borate complex exists as a mixture of several molecular species differing in borate content, molecular weight, and molecular conformation. The solubility varied with pH and was minimal at neutrality. Throughout the pH range it was one to two orders of magnitude higher than that of the parent compound. The molecular size distribution, as determined by differential ultrafiltration, showed a progressive increase in the weight fraction of aggregates going from acid to alkaline solutions. The sizes of aggregates ranged from under 25 to over 100 molecules. The borate content of the complexes increased with increasing pH. No borate was complexed in acid solutions. This indicated that amphotericin B and borate ions can complex to form copolymer chains of varying length in which these species alternate, since both are bifunctional. The complexation equilibrium is favored by high pH. Absorption and CD spectra indicated that the polyene molecules can stack reversibly to form dimers. Dimerization constants calculated from the spectra were highest in neutral solution and declined with increasing acidity or alkalinity. In alkaline solutions the polymer chains are long and extended, with minimal stacking. In neutral solution the chains are shorter and extensively stacked. In acid solutions no borate complexes are formed, and the polyenes are stacked to an intermediate degree. The very different effects of pH and concentration on the degree of complexation with borate and on the degree of dimerization of the polyenes shows that these equilibria are independent of each other.

Notizen: A major component of pigment gallstones (PS) is a black, insoluble substance. It has been suggested that this pigment material might be a highly crosslinked polymer, and if such were the case, it should imbibe solvent (swell) to the maximum permitted by the crosslinks of its macromolecular network. We measured the equilibrium amount, qeq, by which pulverized, desiccated PS swells in different liquids, including isotonic aqueous buffers at pH values from 1.5-11.5. For ionic strengths ≥ 0.15, the dependence of qeq on pH exhibits a broad titration curve with a midpoint near pH 7. qeq was 〈 1.2 in methanol, dimethylformamide, dimethylsulfoxide, and chloroform. The ir absorbance from vinyl groups in the black pigment was only one-eighth that of unconjugated bilirubin, the primary chemical building block of PS; this implicates vinyl groups in the formation of a polymer network. The rise in qeq with increasing pH suggests that the carboxyl groups are free to ionize and are therefore not involved in the covalent bonds that make the crosslinked polymer. A network polymer structure would account for the inability to dissolve PS in those solvents in which unconjugated bilirubin is soluble.

Notizen: 25Mg-nmr data are reported that address the nature of the magnesium ion-DNA interaction. It is found that competitor ions such as calcium, mercury, zinc, and cobalt ions are not effective in competing for all of the magnesium ion-DNA interaction that is reported by the 25Mg-nmr spectrum. The temperature dependence of the 25Mg-nmr spectrum in DNA solution studied at high concentrations of competitor ion indicates that the chemical-exchange lifetime of the magnesium ions at DNA binding sites makes a major contribution to the 25Mg-nmr line width. However, the activation parameters are not consistent with the temperature dependence of either transport properties or chemical exchange with phosphate groups alone, but are consistent with a sum of at least two processes that provide opposing contributions to the 25Mg-nmr relaxation. It is also shown that the non-Lorentzian character of the 25Mg-nmr line previously reported is consistent with the effect of an incompletely averaged static nuclear electric quadrupole interaction and/or an exchange process that is slow with respect to the magnitude of this interaction. Because the concentrations employed in these experiments are high, the present data do not provide a direct or critical test of the electrostatic theories of ion-polyelectrolyte interaction. The present data do demonstrate, however, that such theories alone are insufficient as a basis for understanding the 25Mg-nmr data.

Notizen: The persistence length of lugworm cuticle collagen in 0.1M acetic acid was evaluated as 1600 ∼ 1800 Å by Yamakawa-Fujii's model for a wormlike chain from the sedimentation constant and the intrinsic viscosity. The persistence length was further examined for a series of sample “collagen sonicates” produced by varying the duration of sonic irradiation. To estimate the salt effect on the persistence length, measurements were made over a range of NaCl concentrations from 0 to 0.1M. The results showed that the cuticle collagen and collagen sonicates had identical values of persistence length and that the neutral salt effect for the cuticle collagen was far smaller than that for DNA.

Notizen: We have used translational diffusion coefficient measurements and subunit hydrodynamic theory to determine the dimensions and shape of bacterioophage T4D baseplates and tails. The diffusion coefficient of the baseplate, measured by quasielastic laser light scattering (QLS), was determined previously by Wagenknecht and Bloomfield to be D = 8.56 × 10-8 cm2/s. For the tail, we found D = 5.88 × 10-8 cm2/s by QLS, and D = 6.02 × 10-8 cm2/s by combining sedimentation coefficient and molecular weight in the Svedberg equation. These values, which have an uncertainty of ±2.7%, when combined with subunit hydrodynamic theory, enabled us to refine estimates of dimensions obtained by electron microscopy. For the hexagonal baseplate, the vertex-to-vertex distance is about 480 Å, the thickness is 160 Å, and there are six extended short fibers 320-Å long and 40 Å in diameter. When a baseplate of these dimensions is attached to a tail tube-sheath-connector complex 1050-Å long and 240 Å in diameter, the calculated D is 5.93 × 10-8 cm2/s, within 1% of experiment. This combined use of electron microscopy and hydrodynamics, using the former to ascertain shape, and the latter to obtain solution dimensions, is a powerful approach to the structure of biomolecular complexes.

Notizen: Poly(L-lysine) of various molecular weights between 2700 and 475,000 was spin-labeled. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined by calculation of the rotational correlation time as the poly(L-lysine) underwent the pH-induced random coil to α-helix conformational transition. In general, the rotational correlation time of the nitroxide increased as the pH was increased, indicating a more restricted environment for the spin label when poly(L-lysine) is deprotonated. For the high-molecular-weight poly(L-lysine) this corresponds to the formation of the α-helix and indicates that the side chain-side chain interaction and decreased segmental motion of the backbone (slightly) restricts the motion of the spin label. For the 2700-molecular-weight poly(L-lysine), previously shown not to assume a helical conformation at high pH, the increase in the rotational correlation time of the spin label indicates that the side chain-side chain interaction takes place after deprotonation but without helix formation. This may indicate that helix formation per se is not needed to produce the observed effect even with the high-molecular-weight polymers. The rotational correlation time of the spin label at a particular pH did not depend on the molecular weight of the poly(L-lysine) over the 200-fold range of molecular weights. This indicates that the rotational correlation time reflects the rotational mobility of the spin label in a localized environment and not the rotational diffusion of the entire macromolecule.

Notizen: The interaction of fluoroquinacrine, 3-fluoro-7-chloro-9-(diethylamino-1-methylbutyl-amino)acridine, with poly(A), DNA, and tRNA has been investigated by monitoring changes in the 19F-nmr properties, the fluorescence, and the optical absorbance of the drug. The changes in the properties of fluoroquinacrine in the presence of nucleic acids are similar to those observed for quinacrine and suggest that the drugs bind in a similar fashion. The molecular dynamics of fluoroquinacrine bound to nucleic acids were determined by interpreting the data from a number of different nmr relaxation experiments with a two-correlation-time model. The two motions are the long-range bending motion of the drug-nucleic acid complex and the sliding of the drug between the base pairs. Both dipolar and chemical shift anisotropy contributions to the nmr relaxation parameters were taken into consideration. The binding of fluoroquinacrine to tRNA appears to be different from that observed for binding to DNA. Optical absorbance and 19F-nmr were also used to examine the helix-to-coil transitions of the drug-nucleic acid complexes. In the DNA complex, the 19F chemical shift changes parallel the absorption changes that occur during the transition. 19F-nmr and absorption show that the drug-tRNA complexes undergo a cooperative helix-to-coil transition, with the drug binding sites melting when the tRNA is 70% denatured.

Notizen: The crystal structure of the title compound, a model for the glycosyl linkage between the asparagine side chain and N-acetyl glucosamine in glycoproteins, has been determined and compared to other model structures. The pyranose ring in the crystal is in the 4C1 chair conformation and the amide functions at C1 and at C2 are both oriented such that the amide protons are nearly trans to their respective sugar-ring protons. Coupling constants determined from the fully assigned proton nmr spectrum in aqueous solution are consistent with the conformation in the crystal.

Notizen: Flow linear dichroic studies have been conducted on phage T4B in its fast and slow forms. The behavior of the phages is well represented by an equivalent ellipsoid model using the Peterlin-Stuart theory. The measurements permit the evaluation of the optical factor of the DNA in the phage and the rotary diffusion coefficient of the phage particle. Both these quantities change during the slow-fast conversion. The rotary diffusion results are in good agreement with those obtained by other workers with other methods. The optical factor is negative, indicating a net alignment of DNA helices parallel to the phage axis. The results exclude certain simplified models for the packaged DNA but do not lead to a unique structural conclusion. The flow dichroism experiment and its interpretation are described, and a simple method of calculating optical factors for complicated but cylindrically symmetric structures is presented.

Notizen: It has recently been proposed that the repeating backbone nucleotide may be regarded as consisting of two blocks of equal magnitude representable by two virtual bonds. Implicit consideration of the nucleotide (ψ,ψ) and internucleotide (ω′,ω) geometry that generate variety in polynucleotide conformations, and of the constancy of the repeating structural moieties (P-C4′ and C4′-P) independent of the above rotations, has enabled us to utilize this scheme in the study of ordered structures such as di-, oligonucleotides and, most significantly, tRNA. The polynucleotide folding dictated by short-, intermediate-, and long-range interactions in the monoclinic and orthorhombic forms is described and compared through circular plots depicting the virtual bond torsions and distance plots constructed independently for backbone as well as bases. The torsions and the bond angles associated with the virtual bonds afford a clear distinction between ordered helical segments from loops and bends of tRNA. Lower virtual bond torsions (-60° to 60°) concomitant with higher values of virtual bond angles characterize various bend regions, while torsions around 160°-210° typify ordered helical strands. The distance plot elucidates the type of interaction associated with various sub-structures (helix-helix, helix-loop, and loop-loop) that form the constituents of different structural domains. Several other features such as the manifestation of the P10 loop and the approximate twofold symmetry in the tRNA molecule are conspicuous on the distance plot.

Notizen: The probability of DNA base-pair opening was calculated at temperatures below the denaturation region. The helix-coil transition theory, modified to include different nearest-neighbor interactions, was employed. Predictions of base-pair opening employing DNA melting-curve parameters differed considerably from predictions based on parameters evaluated from synthetic RNA oligomer data of Gralla and Crothers (G-C) and formaldehyde-DNA binding experiments of McGhee and von Hippel (M-vH). Calculations based on the latter parameters indicate a base-pair-opening probability of 10-2-10-3 at 35°C in 0.1M NaCl. DNA melting-curve parameters predict values about 103 smaller. At a temperature 10°C below the transition midpoint of a specific DNA sequence, DNA melting parameters predict base-pair opening of about 10-3, whereas the G-C and M-vH parameters predict that ≳0.16 of the DNA is melted. Experiments and theoretical assumptions relevant to the calculations are analyzed. Evidence suggests that DNA melting parameters are valid when the average loop size exceeds some minimum value, whereas the G-C and M-vH parameters appear more valid for single base-pair loops. A reconciliation of the two sets of predictions can be made if interactions extending beyond neighboring base pairs are considered. Such interactions will make the parameters of the nearest-neighbor model appear to change with the average loop size. Experiments that may provide further measurements of base-pair opening are discussed.

Notizen: Starting with β-methoxy methacryloylisocyanate, β-methoxy methacrylisothiocyanate, and β-isocyanatopropionyl chloride, on the one hand, and Nα-Z-lysine or Nα-Z-ornithine, on the other hand, Nα-Z-amino acids with pyrimidine bases in the side chain were synthesized. These Z-protected nucleoamino acids were converted to the corresponding N-carboxyanhydrides (NCAs) via the silylester method. In the case of 2-thiothymine derivatives, the reaction intermediate of the NCA synthesis caused benzylation of the thioxo- group, so that a new class of 2-mercaptopyrimidine derivatives was isolated unexpectedly. The poly(nucleoamino acids) obtained by polymerization of the nucleoamino acid NCAs were characterized by elemental analyses, optical rotations 1H-nmr and 13C-nmr spectra. Vapor pressure osmometry revealed that the DPs were in the range of 20-30. Their spectra suggest a helical secondary structure. While all homopolypeptides are insoluble in water, copolypeptides containing L-lysine Nε-hydrobromide possess good solubility in water.

Notizen: Vibrational circular dichroism (VCD) spectra for the principal amide stretching vibrations, amide A (N—H stretch) and amide I (predominantly C=O stretch), are presented and analyzed for a variety of polypeptides dissolved in chloroform, as well as for two examples in D2O. Our results for poly(γ-benzyl-L-glutamate) confirm the first and only previous report of VCD in polypeptides carried out by Singh and Keiderling [(1981) Biopolymers 20, 237-240]. Collectively, our spectra show that the sense of the bisignate VCD in these two regions depends on the sense of α-helicity and not on the absolute configuration of the constituent amino acids. This conclusion is established by obtaining VCD for the two polypeptides, poly(β-benzyl-L-asparate) and poly(im-benzyl-L-histidine), that form left-handed as opposed to right-handed α-helices. A new amide band having significant VCD intensity owing to its Fermi resonance interaction with the N—H stretching mode has been identified as a weak shoulder on the low-frequency side of the amide A band near 3200 cm-1 and is assigned as a combination band of the amide I and amide II vibrations. VCD spectra of polypeptides in D2O solution, although weak, have been successfully measured in the amide I region, where spectra appear to be more complicated due to the presence of solvated and internally hydrogen-bonded amide groups. Strong monosignate contributions to the VCD in the amide A and amide I regions for some of the polypeptides indicate coupling of an electronic nature between these two regions and is deduced by an application of the concept of local sum rules of rotational strength. It appears that a detailed understanding of the VCD obtained for polypeptides will not only be diagnostic of secondary structure, but also of more subtle structural and vibrational effects that give rise to local, intrinsic chirality in the amide vibrations.

Notizen: A theoretical analysis of the binding curves for the bifunctional peptide antibiotic echinomycin and its analogs interacting with a variety of DNAs is presented. The method is an extension of our previous work using the sequence-generating-function technique and has been modified to allow for consideration of cooperativity within the framework of the neighbor-exclusion rule. Binding by both single and double intercalation is included, and the results are, in many cases, superior to the analysis based on single-mode binding. In all cases the data are consistent with the neighbor-exclusion principle for intercalation when analyzed with this approach, while data analysis, using a simpler method, often led to violation of this rule. Positive cooperativity is considered in the treatment of data for binding to poly(dA-dT). Finally, we discuss the relationship between base specificity and binding site size.

Notizen: Quasielastic light scattering and electrophoretic light scattering studies were carried out on mononucleosome and oligonucleosome systems. The electrophoretic light scattering experiments employed static and sinusoidal electric fields. Data are presented that suggest at least two relaxation modes. It is proposed that the small amplitude sinusoidal field effectively polarizes the ion atmosphere about the polyion, thus leading to an induced dipole moment that varies sinusoidally in time. This model is, in essence, an extension of the current interpretation of low-frequency dielectric dispersion data on DNA as being due to fluctuations of counterions along the polyion.

Notizen: Human fibrinogen was treated with thrombin in the presence of fibrinoligase (Factor XIIIa) and calcium ion at pH 8.5, ionic strength 0.45, and the ensuing polymerization was interrupted at various time intervals (t) both before and after the clotting time (tc) by solubilization with a solution of sodium dodecylsulfate and urea. Aliquots of the solubilized protein were subjected to gel electrophoresis on polyacrylamide gels after disulfide reduction by dithiothreitol and on agarose gels without reduction. The degree of γ-γ ligation was determined from the former and the size distribution of ligated oligomers, for degree of polymerization x from 1 to 10, from the latter. In some experiments, thrombin was inhibited, after partial polymerization, by p-nitrophenyl-p′-guanidinobenzoate. From these, it was concluded that for thrombin concentration ≤0.013 units/mL and fibrinoligase ≥30 mg/L, oligomer assembly is rapid compared with peptide A release and ligation is rapid compared with assembly. Under these conditions, the theory of the first paper of this series describes rather well the time dependences of the degree of γ-γ ligation, the weight fractions of monomer and small oligomers, and the number- and weight-average degrees of polymerization after solubilization of the staggered overlapped assemblies, each of which splits to give two strands of end-to-end ligated oligomers. The theory assumes that the second A peptide is released by thrombin more rapidly than the first by a factor q, which, from the experimental data, is determined to be 16. The subsequent assembly into staggered overlapped oligomers follows the statistics of linear polycondesation taking into account the presence of both difunctional and monofunctional combining units. For higher thrombin or lower fibrinoligase concentrations, ligation fails to keep pace with oligomer assembly, and the size distributions after solubilization show a higher proportion of very small and a lower proportion of larger ligated oligomers, owing to separation of the staggered overlapped assemblies into smaller fragments.

Notizen: A theoretical model to determine the probability of loop formation, based on an elaborated form of the Jacobson-Stockmayer theory of cyclization equilibria, has been developed and used on RNA chains of homogeneous puckering and lengths up to 27 residues. The probability Qx (q, γ0, ε0) of occurrence of hairpin loops of a particular chain x is given by Qx (q, γ0, ε0) = [W (q) δr][2Γq (γ0)δγ] [Eq,γ0 (ε0) δε] where W(q) is the three-dimensional density distribution function of end-to-end vectors r centered about the ideal loop closure position q; Γq(γ0) is an angular correlation factor that restricts the orientation of terminal bonds (i.e., bond x and bond 1) in the loop to a specified value Δθ = cos-1 (γ0) when r adopts a value within a range δr from position q; and Eq,γ0(ε0) is the probability that ε falls within δε of ε0 when r and γ assume the values denoted by the respective subscripts. The parameter ε is related to the angle between a hypothetical bond (x + 1) and bond 1. In these calculations, Eqγ0(ε0) was set equal to 1. For randomly coiling models previously developed to reproduce polynucleotide unperturbed dimensions, loop closure probability is maximized with chains of length 22 residues. Larger hairpin loops of 24-25 residues, also favored in C3′-endo random coils of this type, are potential models of tRNA unfolding. W(q) is a stronger determinant of loop closure than Γq(γ0). The angular correlation effect is most noticeable at chain lengths 22 and 23 where 2Γq(γ0) deviates from unity.

Notizen: High-resolution neutron time-of-flight (TOF) spectra were measured for partly deuterated hen egg-white lysozyme in solution with and without N-acetyl-glucosamine inhibitor bound to the cleft, and in polycrystalline form. Weak but reproducible bands occur at frequencies between 25 and 375 cm-1. The bands were tentatively assigned on the basis of previous results for homopolypeptides. At very small energy transfers (between about 1 cm-1 and 40 cm-1), the TOF spectra show a dependence both on inhibitor binding and crystalline environment. This is interpreted in terms of conformational flexibility.

Notizen: Transient electric dichroism has been measured for the ferriheme-poly(L-lysine)[(Lys)n], ferroheme-(Lys)n, and ferroheme-(Lys)n-carbon monoxide (CO) solutions at pH 9-12. The Soret absorption maximum in electronic spectrum (λ), the reduced linear dichroism (ρ∞) at complete orientation and the calculated angle (φ) between the porphyrin plane of a bound heme and the oriented polymer axis are determined for the following complexes: a ferriheme-(Lys)n complex at pH 9.5-10.5 (λ = 420 nm, ρ∞ = 0.50, and φ = 19°), a ferroheme-(Lys)n complex at pH 9.5-10.2 (λ = 432 nm, ρ∞ = 0.77, and φ = 0°), and a ferroheme-(Lys)n-CO complex at pH 9.25 (λ = 430 nm, ρ∞ = 0.38, and φ = 24°). Based on the above data, the validity of the structures of heme-(Lys)n complexes proposed by other investigators is discussed.

Notizen: The stability of the α-helices of isomeric block copolypeptides is nonequivalent, as reported previously. In order to explore the origin of the nonequivalence, the stability of α-helix of two block copolypeptides, (L-Ala)20-(L-Glu)20-(L-Phe) (designated as AEF) and (L-Glu)20-(L-Ala)20-(L-Phe) (EAF), in aqueous solution was investigated as a function of pH, temperature, and salt concentration by the measurement of the α-helical content using CD at 223 nm. The transition temperature, Tm, as a measure of the stability of the α-helix, decreased with increasing the salt concentration for EAF, while Tm increased for AEF. The results indicate that electrostatic interactions affect the nonequivalence of such helical stability. Thermodynamic quantities, ΔG, ΔH, and ΔS, of the thermal transition from random coil to α-helix were obtained by applying the curve-fitting method to the data. The major contribution to the effects of salts seems to be the entropic term, not the enthalpy term. This is unexpected, since the salt ions would weaken electrostatic interactions between ionized groups and the dipole along the helical axis, which affect the enthalpy term. In addition, the dependence of the electrostatic effect on the salt concentration is different for EAF and AEF. There fore, the nonequivalence cannot be accounted for by only the electrostatic effect, suggesting that it originates from some intrinsic property of the α-helix.

Notizen: Using quasielastic light scattering, we have determined the diffusion coefficients of low-density lipoprotein (LDL) and polystyrene latex spheres over the temperature range of 293-318 K. The results show that after correction for thermal changes in the solvent, there remains a residual negative temperature coefficient in the diffusion amounting to about -0.6%/K that is independent of the chemical type of complex. Confirmation of these results was obtained for LDL through sedimentation studies over a similar range of temperatures. The residual temperature coefficient was similar to that reported for oxyhemoglobin [W. B. Veldkamp and R. Votano (1980) Biopolymers 19, 111-124] and greater than that found for bovine serum albumin by these and earlier workers. These observations show that the residual dependence on temperature is not an isolated phenomenon and could in part be explained by increased aggregation of particles, although this is not the primary cause of the effect.

Notizen: The conformational changes of poly(dA-dT) from random coil to ordered structure with stacked bases produce important changes in the Raman line intensities (hypochromism) when the polymer is excited under the preresonance Raman conditions (λ excitation = 300 nm). Poly(dA-dT)-RNase and poly(dA-dT)-histone H1 interactions have been studied as models of mechanisms of destabilization and stabilization by proteins of the DNA secondary structure, respectively, following this intense preresonance Raman hypochromism. In addition, the specific variation of the intensity of the 1582-cm-1 line of adenine is interpreted in terms of the interaction of the amino group with the RNase (thus involving the large groove). In the poly(dA-dT)-H1 complex, the intensity of the 1665-cm-1 line of thymine increases. This increase appears to involve the C2=O group of thymine, located in the narrow groove.

Notizen: The enthalpies of binding of chloroquine and quinacrine to DNA at different molar ratios of drug to DNA and at different ionic strengths have been measured. The limiting values obtained with quinacrine fall in the range found for typical intercalating agents (e.g., ethidium, proflavin, adriamycin), whereas the value obtained with chloroquine is always zero, independent of the ratio of drug to DNA and ionic strength. The dilatometric measurements performed on the same systems and on the ethidium-DNA system show that when ethidium and quinacrine bind to DNA at low drug/DNA ratios, a volume decrease of about 16 mL/mol of bound drug occurs. No change in volume is observed when the two drugs bind to DNA through external, electrostatic forces. The volume change can be attributed to the loss of structured water around hydrophobic moieties of the drug molecules, following intercalation. In contrast, chloroquine binding to DNA at low drug/DNA ratios is characterized by a volume change distinctly smaller than that shown by quinacrine. The low ΔVB and ΔHB values shown by chloroquine are discussed in terms of the mechanism of interaction with DNA.

Notizen: The conformation and the dynamic structure of single-stranded poly(U) and poly(C) in neutral aqueous solution have been investigated by 1H-nmr at two different frequencies (90 and 250 MHz) and at various temperatures. Measurements of proton chemical shifts, coupling constants JH-H, and proton relaxation times, T1, T2, versus temperature show a striking difference in conformation and in dynamic structure between the two polynucleotides studied. The temperature effect on δ and JH-H is found to be substantial for poly(C) and insignificant for poly(U). The S conformer is favored in poly(U), whereas the N conformer strongly predominates in poly(C) (≃90%), similar to the case for RNAs. These results suggest that single-stranded poly(C) probably possesses a helical or partial helical structure, whereas poly(U) shows a clear preference for the random coil, in agreement with the optical results. The local motions of the ribose and base were studied at various temperatures by measurements on the relaxation times at 90 and 250 MHz. For a given temperature between 22 and 72°C, the ratio T1(90)/T1(250) is practically the same for all poly(U) protons, indicating that in this temperature interval the ribose base unit of poly(U) undergoes an isotropic motion characterized by a single correlation time τc. Above 52°C, poly(C) exhibits a dynamic structure similar to poly(U). Below this temperature, poly(C) exists in an equilibrium between randomly coiled and single-stranded helix forms. This situation is characterized by a strong cross-relaxation effect and T1 values corresponding to a relatively short apparent correlation time. An activation energy of 4 kcal/mol was determined for the motion of the ribose-base unit in both single-stranded polynucleotides.

Notizen: The complexes of the three BrCN-cleaved fragments of sperm whale apomyoglobin with hemin were studied by circular dichroism (CD). In native myoglobin, the heme is located in the middle fragment; the isolated peptide (residues 56-131), however, produces little extrinsic Cotton effects by the addition of hemin, although about four molecules of hemin are bound to this peptide. In marked contrast, the COOH-terminal peptide (residues 132-153), which binds three hemin molecules, shows strong Cotton effects in the Soret bands and drastically changes its conformation from unordered to highly helical. The Arg-modified or Lys-deaminated peptide no longer undergoes conformational changes by the addition of hemin, suggesting that the two propionic acid groups of one hemin molecule interact with the Arg residue and one of the Lys residues, which stabilizes the induced helical conformation. The NH2-terminal peptide (residues 1-55) binds one hemin molecules, and the helicity of this fragment is slightly enhanced by the addition of hemin. Both the CD and difference absorption spectra indicate that the mode of interaction between the peptides and hemin are different for the three apomyoglobin fragments.

Notizen: CD study of four modified nucleosides, constituents of tRNA molecules, revealed that 2-thio-5-methyluridine and 5-methyluridine in aqueous solution, 0.1N HCl, and organic solvents essentially occur in an anti-conformation. 5-Methylcytidine also occurs in an anti-conformation similar to cytidine in aqueous solution and organic solvents, while 2-thiocytidine dihydrate appears to occur in an anti-conformation. It is stressed that the CD data of thionucleosides might be applied to the successfully conformational analysis of tRNA molecules.

Notizen: It has been inferred from previous studies that Mn2+ ions bind preferentially to G·C base pairs in DNA, and it has even been suggested that this preference for G·C pairs might be responsible for some of the Mn2+ specific effects observed in various biochemical reactions. In this paper we investigate the AT/GC preference of Mn2+ by direct competition studies in which AT-rich DNA was dialyzed against GC-rich DNA in the presence of varying amounts of Mn2+. Analysis of these results demonstrates that over a wide range of Mn2+/DNA(P) molar ratios, Mn2+ binds to A·T and to G·C base pairs with virtually identical affinity, although in a somewhat different mode. Both the present and previous nmr, uv, CD, and melting studies are discussed in terms of the different modes of binding of Mn2+ to single- and double-stranded DNA.

Notizen: We have determined extinction angles and flow birefringence of T7 bacteriophage DNA over a wide range of shear, polymer concentration, and solvent ionic strength. From these data, information on the simple salt dependence of coil permeability to solvent and on short-range intrachain interactions (persistence length) was obtained. At all ionic strengths, our results are consistent with a partially draining coil in the Gaussian subchain dynamical theory of Rouse-Zimm-Tschoegl-Bloomfield. Salt dependence of persistence length is comparable to, although somewhat less than, that obtained previously using similar methods with a fivefold higher-molecular-weight DNA (T2 bacteriophage DNA). Possible reasons for observed discrepancies are analyzed, and the results of this work are compared in detail to other current studies of solvent ionic strength dependence in persistence length and hydrodynamic properties of DNA.

Notizen: Variation of collagen fibril structure in tendon was investigated by x-ray diffraction. Anatomically distinct tendons from single species, as well as tendons from different species, were examined to determine the variations that exist in both the axial and lateral structure of the collagen fibrils. The meridional diffraction is derived from the axial collagen fibril structure. Anatomically distinct tendons of a particular species give meridional patterns that are indistinguishable within experimental error. The meridional diffraction patterns from tendons of different mammals are similar but show small species-specific variations, most noticeably in the 14th-18th orders. Tendons of birds also give meridional patterns that are similar to each other, but the avian patterns differ considerably from the mammalian ones. Avian tendons give stronger odd and weaker even low orders, a feature consistent with a reduced gap:overlap ratio, and have a distinctive intensity pattern for the higher meridional orders. Interpretation of these differences has been approached using biochemical data, diffraction by reconsituted fibers of purified collagen, and Fourier transform analysis. From these methods, it appears that the variations observed in the lower orders (2nd-8th) and in the higher orders (29th-52nd) are probably related to differences in the primary structure of the Type I collagen found in the different species. The variations observed in the 14th-18th orders appear not to be related to features within the triple-helical domain of the molecule.Equatorial diffraction yields information on the lateral packing of collagen molecules in the fibrils, and considerable variation was seen in different tendons. Rat tail tendon gives sharp Bragg reflections, demonstrating the presence of a crystalline lateral arrangement of molecules in the fibril. For the first time, sharp lattice reflections similar to those in rat tail tendon have been observed in nontail tendons, including rat achilles tendon, rabbit leg tendon, and wing and leg tendons of quail. In the rabbit and quail tendons, one of the strong equatorial reflections characteristic of the rat tendon pattern, at 1.26 nm, was absent. The positions of the equatorial maxima, which are a measure of intermolecular spacing, varied considerably, being smallest in the specimens displaying crystalline packing. The intermolecular distance in chiken and turkey leg tendons is longer than that found in mammalian tendons, or in avian wing tendons, which supports the hypothesis that a larger intermolecular spacing is characteristic of tendons that calcify.Thus, x-ray diffraction indicates there are reproducible differences in both the axial and lateral structure of collagen fibrils among different tendons. This work on tendon, a tissue containing almost exclusively Type I collagen as its major component, should serve as a basis for analyzing the structure of other connective tissues, which contain different genetic types of collagen and larger amounts of noncollagenous components.

Notizen: The conformation of poly(α-L-aspartic acid) was investigated on a sample in which β-bonds were not detected. CD and ir spectroscopy showed that poly(α-L-aspartic acid) passes through a conformational change induced by changes of the degree of ionization that is accompanied by precipitation; the precipitate is probably highly helical. The change was also detected by potentiometric titration.

Notizen: We studied the kinetics of the heat denaturation (at 50°C) of met-hemoglobin in the presence of various monohydric alcohols. The denaturation rate was slowed by the presence of small concentrations of methanol and ethanol; in all the other cases, i.e., at high concentrations of methanol and ethanol, and in the whole concentration range studied for iso- and n-propanol, an increase in the denaturation rate was observed. Following a procedure already applied to the study of the effects of the same alcohols on the reaction of hemoglobin with oxygen, we separated the overall observed effects into contributions related to the variation of the bulk dielectric constant of the medium (bulk-electrostatic contributions) and contributions not related to variations in this parameter (nonbulk-electrostatic contributions). For all the alcohols, we found a unique correlation law connecting the above nonbulk-electrostatic contributions with the analogous ones previously reported for the T → R transition of hemoglobin. This fact strongly supports the validity of the procedure used and suggests that nonbulk-electrostatic contributions, relative to these two different processes, have a common background and are univocally determined by the extent of the “solvent perturbation” imposed by the presence of the perturbing cosolvents.

Notizen: 2,2,′,2″,2‴-Tetrapyridineiron(III) complex ions anchored to poly(L-glutamate) (FeL) or poly(D-glutamate) (FeD) were used as catalysts for the H2O2 oxidation of L(+)-ascorbic acid at pH 7 and varying complex:polymer-residue molar ratios [C]/[P]. Evidence is produced that the reaction is a composite process reflecting contributions from parallel pathways, one of which corresponds to a catalytic route and is [H2O2]-independent and the other to an uncatalyzed electron-transfer process between the ascorbate anion and hydrogen peroxide. Stereospecific effects in the catalysis are observed on increasing the complex:polymer ratio, which corresponds to an increase of the amount of α-helical fraction in the polypeptide supports (xa). Thus, at [C]/[P] = 0.01 (xa 〈 0.05), kFeD/kFeL = 1.0; but at [C]/[P] = 0.20 (xa ≈ 0.70), kFeD/kFeL = 4.0 ± 0.5, kFeD and kFeL being the second-order rate constants of the electron-transfer reaction between the FeD or FeL isomer of the asymmetric catalyst and the L-ascorbate anion. The activation energies were found to increase markedly on going from the former to the latter complex:polymer ratio but, at the same time, to exhibit equal values with both enantiomeric catalysts. Stereoselectivity therefore appears to be an entropy-controlled process, arising from the conformational rigidity of the precursor complex, which very likely sees the substrate molecules bound to the chiral residues of the ordered polymer surrounding the active sites. The implications of the stereochemical features of the substrate-catalyst adduct on the mechanism of electron transfer are also discussed. Evidence is presented that the asymmetric [Fe(tetpy)(OH)2]+-polyelectrolyte systems play the additional role of environmental controller of the uncatalyzed oxidation of the L-ascorbate anion.

Notizen: A temperature-jump relaxation study of the interaction of tilorone with different polynucleotides and DNA has been performed. A single relaxation time, attributed to the intercalation step, has been observed in the case of poly[d(A-T)]·poly[d(A-T)], poly[d(A-C)]·poly[d(G-T)], poly[d(G-C)]·poly[d(G-C)], and poly(dG)·poly(dC). No intercalation into poly(dA)·poly(dT) occurs, and the interaction with poly(dG)·poly(dC) is different from what is observed with the other intercalating homopolymers. Refinement of the binding model is suggested from the analysis of the kinetic data. The relaxation curves obtained with DNA are well simulated based on a binding mechanism where DNA is considered a heterogeneous lattice and each type of site behaves as if it were located in the corresponding homopolymer. Poly(dA)·poly(dT) shows a unique behavior: studies of the effects of concentration and temperature indicate that tilorone acts as a probe of a process involving the polynucleotide alone. This process appears to be related to the dynamic structure of the nucleic acid and is detectable only when the bound dye is not intercalated.

Notizen: We had earlier observed that the detailed nature of the β-structure adopted by poly(L-tyrosine) depends on molecular weight [Auer, H. E. & McKnight, R. P. (1978) Biochemistry 17, 2798-2805]. It was proposed that shorter molecules form an open, single-layered sheet (class I) and that longer chains fold over to generate at least two layers (class II). In this work, solvent perturbation difference spectroscopy of the chromophoric side chain was applied to gain additional support for the model. Two sample-reference pairs of media, sodium acetate vs sodium chloride and urea vs buffer, were studied. Great care was taken to achieve identical conditions in the sample and reference cells. The factors of importance in executing the experiments are the polypeptide concentration, the degree of side-chain ionization, achievement of the same class of β-conformation, and the absence of significant aggregation. Difference spectra obtained with both solvent pairs have similar features. Low-molecular-weight samples manifest difference bands arising from both ionized and neutral tyrosine side chains, while high-molecular-weight samples yield spectra with contributions primarily from ionized residues. The interpretation of these results is that the class I particle has both ionized and neutral residues exposed to the solvent medium, whereas the class II particle is folded, with neutral residues restricted to the interior of the fold and primarily ionized residues on the outer surfaces exposed to the solvent. Our results, therefore, corroborate the original model.

Notizen: Extinction angles, flow birefringence, and intrinsic viscosities are compared for linear, bihelical DNAs from viral and other sources that span a range in molecular weight from ∼105 to 1.3 × 108. This range effectively spans the region over which transition from rigid-rod to expanded-coil hydrodynamic property behavior occurs. All DNAs are in identical, phosphate-EDTA, neutral-pH buffers, 0.1M in NaCl. The extinction angle is a hydrodynamic property only and is thus particularly sensitive to effects of kinetic chain rigidity or internal viscosity. Our extinction angle results cannot be interpreted by any simple, single-function theoretical expression. Rather, they must be divided into distinct high- and low-molecular-weight domains. The low-molecular-weight region is typical of rigid-particle opticohydrodynamic property behaviour characterized primarily by particle orientation. The high-molecular-weight domain shows evidence for a finite internal viscosity effect, however, which can be interpreted as very nearly Kuhnian using Cerf's amplification of the Gaussian subchain model to include internal viscosity. It is found that the high-molecular-weight, monodisperse viral DNAs from T7, T5, and T2 bacteriophage show an internal viscosity contribution to the limiting extinction angle-shear rate ratio of ∼3 × 10-3 s. An effect of this magnitude may be marginally important in interpreting extinction angle and certain other hydrodynamic property data for high-molecular-weight DNA systems. Internal viscosity effects do not appear to be manifest in the ratio of flow birefringence to intrinsic viscosity, however, and the persistence length of the high-molecular-weight DNAs is found to be independent of molecular weight to within estimated experimental uncertainty.

Notizen: Phosphorylase kinase contains four approximately equivalent binding sites for 1-anilinonaphthalene-8-sulfonate (1,8-ANS). Measurements of the time decay of fluorescence anisotropy have failed to give any indication of internal degrees of rotational freedom involving a significant portion of the tertiary structure. In the presence of 1 mM Ca2+, calmodulin binds one molecule of 1,8-ANS. No binding occurs in the absence of Ca2+. The binding is strongly temperature-dependent, a decrease in binding occurring with increasing temperature. Determinations of the time decay of fluorescence anisotropy indicate the presence of internal rotations, which become more important with increasing temperature. Complex formation between phosphorylase kinase and calmodulin reduces the binding of 1,8-ANS.

Notizen: The dissociation of beef liver and bacterial (Micrococcus lysodeikticus) catalases by the action of sodium n-dodecyl sulfate (SDS) has been investigated as a function of SDS concetration and time by ultracentrifugation. The rate of dissociation of beef liver catalase is found to be much faster than that for bacterial catalase in 25 mM SDS at pH 7.0. Beef liver catalase is dissociated into its four subunits after 24 h, whereas bacterial catalase is not completely dissociated after 36 days of incubation. The binding of SDS to beef liver catalase obeys a Hill equation with a cooperativity exponent of 2.0 and a binding constant of 440. The initial interaction of SDS with beef liver catalase can be detected by microcalorimetry, whereas the mixing of SDS with bacterial catalase is athermal. Bacterial catalase retains enzymic activity in the presence of SDS, whereas beef liver catalase is completely deactivated at SDS concentrations above 5 mM. Beef liver catalase is more sensitive to acid denaturation than bacterial catalase, and the rate of dissociation for both catalases is sixth-order in proton concentration. Comparison of the amino acid analysis of the two catalases shows that bacterial catalase has a smaller number of lysyl residues and a larger number of glutamyl residues than beef liver catalase. Taken together these structural differences could lead to a reduced affinity of bacterial catalase for the binding of SDS as observed.

Notizen: The Avileurekanoses A and C and Further Degradation Products of the Avilamycins A and CAlkaline hydrolysis of the orthosomycin antibiotics, avilamycins A ad C products similar t those earlier described for flambamycin. Among these avileurekanose c (15) became of particular importance because its acetate prisms suitable for an X-ray structural determination. For the avilamycins A and C structural formulae 2 and 3, respectively, can now be drawn, in which only the configuration at C (16), one out of 32 asymmetric C-atoms, is not yet determined.

Notizen: The complexation of Cue2+ with 1, 8-diamino-3, 6-diaza-2, 7-octanedione (—N, N′-diglycyl-1, 2-ethanediamine, DED) and with 1, 9-diamino-3, 7-diaza-2, 8nonanedione (—N, N′-diglycyl-1, 3-propanediamine, DPD) has been studied by potentiometric and by spectrophotometric titration. With both ligands L the complexation to Cue2+ leads to relatively complicated equilibria with CuLH3+, CuL2+, CuLH-2, and dimeric Cu2L24+ complexes. With DED, another dimeric species, Cu2L2H-22+, is formed in addition. Independent numerical treatment of spectrophotometric and poteritiometric titrations was used to obtain a satisfactory model for the complexation and to test the relative discriminatory power of the two methods. Titrations of glycine ethylamide (GEA) were used as an additional test and as a model for DED and DPD. It was shown that in each case spectrophotometric titrations give results of similar reproducibility and have a discriminatory power equal to or better than potentiometric titrations, provided that optimum mathematical algorithms are used in the numerical treatment.

Notizen: Heterocyclic and Carbocyclic 12-π-and 14-π-Systems, 47th Commnunication1. Synthesis of 7,9-Dimethyl-4,5-dihydro-3H-benz[cd]azualene-3-one and 7,9-Dimethyl-3H-benz[cd]azulene-3-one. A Simple Synthesis of Azulenopseudophenalenons4, 6, 8-Trimethylazulene (3) reacts after metalation with lithiumdiisopropyl-amide in ether with bromoacetic acid to the 6, 8-dimethylaltulene-4-propionic acid (4), which undergoes cyclization to the 7, 9-dimethyl-4, 5-dihydro-3H-benz [cd]-azulene-3-one (5) in the presence of p-toluenesulfonic acid; oxidation of 5 with 2, 3-dichloro-5, 6-dicyanobenzoquinone yields 7, 9-dimethyl-3H-benz [cd]azulene-3--one (1b). Alkylation of 1b with triethyloxonium tetrafluoroborate in CH2C12 gives the 3-ethoxy [cd]benzazulenium tetrafluoroborate (6).

Notizen: Doubts that have recently been expressed [1] about the currently accepted (+)-(R), (-)-(S) [2] absolute configuration of indane-1-carboxylic acid appear to be unwarranted. Our ORD. values for 1-methylindane [12] are in error.

Notizen: Synthesis of [16,16,16,16′,16′,16′ 2H6]LycopeneLycopene having at both ends the deuteriated methyl groups trans to the chain has been synthesized for further biochemical experiments aimed to elucidate the stereochemistry of the cyclization step.Incidentally a gas-chromatographic separation of the isotopisomers 1 and 2g has been observed.

Notizen: The dienone-dianion derivatives 1 react with all types of electrophiles tested (alkyl halide, silyl chloride, ester, ketone, aldehyde, epoxide) to give β, γ-unsaturated carbonyl compounds of type A (see Formulae 2-6, 13, 14 and Tables 1-5). The α- and β-hydroxyalkylation products obtained from 1a-1d can be converted to tetra-hydrofuran and tetrahydropyran derivatives 7 and 16, respectively (Tables 1 and 2), those from the sulfur analogues 1e and 1f to ketene thioacetals 9 and to dienone derivatives 10 and 12. The t-butyl and α-hydroxy-ketones are cleaved to give nitriles, amides, carboxylic acids and esters (Formulae 16-25). The reagents 1 allow to synthesize products with distant functional groups in one step (cf. 1,8-diketones 14 and Formulae 26-30); they correspond to the d5-synthons 31-33; in Table 6, they are compared with other d5-reagents.

Notizen: Methyl-2-acetamido-4,6-di-O-acetyl-3-S-acetyl-2-deoxy-3-thio-α-D-mannopy-ranoside has been synthesized by conversion of methyl 2-amino-2-deoxy-4,6-O-benzylidene-α-D-altropyranoside into the corresponding 3-O-methanesulfony1-2-N-[(methylthio)thiocarbonyl]derivative followed by intramolecular displacement of the 3-O-methanesulfonyloxy group with the (methylthio)thiocarbamoyl group.

Notizen: (Perhalomethylthio)heterocycles XII. Preparation of Perfluoroalkylsulfonylurea Derivatives as well as CCl3-nFnX-Substituted Heterocycles and Their Biological ActivityXI, Mitt., s.[1]In the presence of CF3SO3H (chlorodifluoromethylthio)thiophene 1 reacts with CClF2SCl to give the 2,5-disubstituted thiophene 2a which on oxidation with 30% H2O2-solution yields the corresponding sulfonyl compound 2b · R1SO2NCO adds amines to give R1SO2NHC(O)NHR 3a-g. Some biological properties of these compounds were investigated.

Notizen: The stereochemical outcomes [ratios (1 R, 2 R, 4 R)-isoborneol/(l R, 2 S, 4 R) isborneol or (1R, 2 S, 4 S)-isoborneol(l S, 2 R, 4 S)-borneol and (1 RS, 2 RS, 4 RS)-isoborneol/(l RS, 2 SR, 4 RS)-borneol] in lithium, sodium and potassium/ammonia reductions without a proton source of the enantiomeric [(1 R, 4 R) or (1 S, 4 S)] and racemic (1RS, 4RS) forms of camphor under the same conditions differ strongly. This explains contradictory reports in the literature and, in the case of the reductions with potassium in which pinacol coupling does not compete, proves that the reductions involve bimolecular reactions between two ketyls, or between the ketone and the ketyl or the ketone dianion.