Some theoretical considerations regarding filter disk assays

doi:10.1016/0003-2697(75)90314-0

Analytical Biochemistry

Volume 67, Issue 2, August 1975, Pages 422-427

https://doi.org/10.1016/0003-2697(75)90314-0 Get rights and content

Abstract

The concept of the efficiency of filter disk assays is examined from a theoretical viewpoint. It is shown that the approaches which have been recommended to measure efficiency in such assays are somewhat oversimplified and can lead to errors in data interpretation.

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The high-performance liquid chromatographic retention parameters (k) have been determined for a series of 29 phenothiazines and related drugs. The k values were obtained on a hydrocarbon-bound silica stationary phase, an aminopropyl stationary phase and an aminopropyl phase coated with melanin. Polycratic retention data determined on a hydrocarbonaceous column were extrapolated to 0% of organic modifier in binary aqueous eluent yielding the chromatographic hydrophobicity parameter, log k′_w. Logarithms of capacity factors determined isocratically on the aminopropyl column were subtracted from analogous values obtained with the same column loaded with melanin. The resulting parameter, log k′_m−a, in combination with log k′_w produced a regression equation (correlation coefficient r = 0.9531, significance level p = 10⁻⁶) which could be used to describe drug—melanin binding efficiency, E_B. Theoretical E_B values were calculated by means of the derived equation for the whole series of 29 drugs chromatographed. The efficiency of binding, E_B to synthetic melanin was also determined by an ultrafiltration method for fifteen members of the series. No statistically significant differences were observed between the E_B values calculated using the chromatographic and ultrafiltration approaches. The results indicate that chemometric analysis of the appropriate chromatographic data is a practical method for the evaluation of melanin binding.
Tight-binding complexes of thymidylate synthetase, folate analogs and deoxyribonucleotides
1982, Advances in Enzyme Regulation
Tightly-bound ternary complexes are formed when dTMP synthetase is incubated with deoxyribonucleotides such as FdUMP, dUMP, dTMP, and folate analogs, presumably mediatedy by conformational changes in the enzyme induced by these ligands. The structure of both the folate analog and the nucleotide determines the tightness of binding in the ternary complex. For example, the first order rate constant for dissociation of FdUMP from the dTMP synthetase-FdUMP PteGlu complex is almost 100-fold larger than from the dTMP synthetase-FdUMP-PteGlu₃ complex. The latter had a slower rate of dissociation than did the covalent dTMP synthetast-FdUMP-5,10-CH₄H₄Pte-Glu complex, which had been shown to have a K_d of 10⁻¹¹ M. FdUMP formed tighter complexes than did either dUMP or dTMP when using PteGlu₃, PteGlu, or H₂PteGlu as the folate components, but with 5,8-deazafolate complexes formed with all three nucleotides appeared to have about the same stability.
H₂PteGlu also induced the tight binding of dUMP to dTMP synthetase. A Scatchard plot indicated positive cooperativity in the binding of dUMP to both human and bacterial enzyme in the presence of H₂PteGlu. These results suggest the possibility that when cells are exposed to MTX the decrease in dTMP synthetase activity may be due largely to formation of inhibitory enzyme-dUMP-H₂PteGlu_n complexes and does not result primarily from depletion of reduced folates because of dihydrofolate reductase inhibition. Because this complex has a slow rate of dissociation, dTMP synthetase activity would still be inhibited for some time after an influx of reduced folates (as in leucovorin rescue) sufficient to support the enzyme reaction under normal conditions.
Equilibrium and kinetic characteristics of steroid interactions with human plasma sex steroid binding protein
1980, Archives of Biochemistry and Biophysics
The kinetic and equilibrium binding parameters for the molecular interaction of testosterone and human plasma sex steroid binding protein have been determined at varying pHs. The second-order association rate constant and first-order dissociation rate constant, at 4 °C, were 1.05 × 10⁸m⁻¹ min⁻¹ and 0.11 min⁻¹, respectively. The equilibrium constant was 1.3 ± 0.1 × 10⁹m⁻¹. All three constants were not significantly dependent on pH in the range of pH 6 to 11. Activation energy for the dissociation reaction was 16.4 kcal · mol⁻¹ and was constant from 4 to 37.5 °C. A series of steroidal and nonsteroidal compounds were tested for their sex steroid binding affinity relative to testosterone. These results all support a description of the steroid binding site in human sex steroid binding protein as hydrophobic, nonionizing (at 5.5 ≤ pH ≤ 11.0), and with high structural specificity.
A flow dialysis technique for the measurement of free testosterone in human plasma under physiologic conditions
1977, Biochemical medicine
A technique is described for measuring the percentage of free (unbound) testosterone under physiologic conditions (37°C in whole plasma) without disturbance of the equilibrium state. The technique employs 10 ml of whole plasma with a tracer amount of tritiated testosterone in a flow dialysis cell. The precision of the technique is 5–10%. The percentage of free testosterone values so obtained for several samples of human plasma are in general agreement with previous estimates. The technique permits re-evaluation of individual plasma samples several times, and it can be easily generalized to the study of other steroids in whole blood or plasma.
The free testosterone is generally believed to be the biologically active portion of the plasma testosterone (1, 2). Free testosterone is that portion of the total which is not bound to plasma binding species. Practically speaking, the free testosterone fraction is that portion of testosterone in plasma which is capable of diffusing through a dialysis membrane. A technique has been described by Colowick and Womack (3) for the measurement of the percentage of diffusible molecules at equilibrium without significantly disturbing the equilibrium state. An apparatus is described here which is a modification of that used by Colowick and Womack and which permits measurement of the percentage of free steroid in plasma under physiologic conditions.
A rapid technique to evaluate two high-affinity binding proteins in the same preparation
1976, Analytical Biochemistry
A modified DEAE-cellulose filtration assay was developed to estimate the capacities of two types of high-affinity progesterone-binding sites. The method was tested using a mixture containing rat corticosteroid-binding globulin and progesterone-binding protein from pregnant guinea pig plasma and by simulation. The assay has proved useful in studying the progesterone-binding proteins in uterine cytosol of the rat and other species and in following the separation of the two types of binding sites in rat uterine cytosol by ammonium sulfate fractionation or differential hydroxylapatite adsorption.
Position statement: Utility, limitations, and pitfalls in measuring testosterone: An endocrine society position statement
2007, Journal of Clinical Endocrinology and Metabolism
The objective of the study was to evaluate the current state of clinical assays for total and free testosterone.
The five participants were appointed by the Council of The Endocrine Society and charged with attaining the objective using published data and expert opinion.
Data were gleaned from published sources via online databases (principally PubMed, Ovid MEDLINE, Google Scholar), the College of American Pathologists, and the clinical and laboratory experiences of the participants.
The statement was an effort of the committee and was reviewed in detail by each member. The Council of The Endocrine Society reviewed a late draft and made specific recommendations.
Laboratory proficiency testing should be based on the ability to measure accurately and precisely samples containing known concentrations of testosterone, not only on agreement with others using the same method. When such standardization is in place, normative values for total and free testosterone should be established for both genders and children, taking into account the many variables that influence serum testosterone concentration.

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Some theoretical considerations regarding filter disk assays

Abstract

J. Mol. Biol

Anal. Biochem