Recent developments in quantitative affinity chromatography

This review surveys developments during the past decade in the use of quantitative affinity chromatography as a means of evaluating equilibrium constants for solute-ligand and solute-matrix interactions. Topics include allowance for multivalency of the partitioning solute, removal of the myth that highly substituted affinity matrices are unsuitable for zonal quantitative affinity chromatography, adaptation of the technique to allow characterization of high-affinity interactions and the application of quantitative affinity chromatography theory to the characterization of biospecific adsorption phenomena in cellular systems

Evaluation of antigen-antibody affinity constants by partition equilibrium studies with a two-phase aqueous polymer system: a more rigorous analysis

A quantitative procedure is devised for the determination of affinity constants from the composition-dependence of the antigen partition coefficient in liquid-liquid partition studies of antigen-antibody mixtures. Its use is illustrated by application to published results (Elling, L.R., Kula, M.-R., Hadas, E. and Katchalski-Katzir, E. (1991) Anal. Biochem. 192, 74-77) obtained for the interaction of horseradish peroxidase with an elicited monoclonal antibody by antigen distribution studies with a two-phase aqueous polymer system (poly(ethylene glycol)/dextran) for the partition analysis. Shortcomings in the original interpretation of these results are eliminated

Measurement of binding constants by capillary electrophoresis

The assumptions inherent in a capillary electrophoresis procedure for evaluating binding constants for interactions between lectins and charged polysaccharides [S. Honda et al., J. Chromatogr., 597 (1992) 377] have been reappraised. Whereas the results were originally interpreted on the basis that the lectin-carbohydrate interaction was restricted to 1:1 complex formation, a more plausible interpretation is shown to be that an approximately constant incremental difference separates the mobilities of the successive complexes formed as the result of saccharide binding to equivalent and independent sites on the lectin. The parameter that is determined by capillary electrophoresis should thus be regarded as the intrinsic binding constant

Evaluation of Cross-Linking and Scission Yields in Irradiated Polymers from the Dose Dependence of the Weight- and z-Average Molecular Weights

A procedure has been developed for the determination of scission and cross-linking fields, G(S) and G(X), in irradiated polymers by combination of the ordinate intercepts of plots of ([Mw(0)/ MW(D)] - 1)/D and ([Mz(0)/A/2(D)] - 1)/D versus dose (D). The required weight- and z-average molecular weight data for a given sample can be obtained by using the Rayleigh and schlieren optical systems, respectively, to record the solute distribution in a single sedimentation equilibrium experiment. The procedure has been tested by application to simulated data for a range of initial molecular weight distributions and values of G(S)/G(X) and applied to previous sedimentation equilibrium results (Nichol, J. M.; Donnell, J. H.; Rahman, NLP.; Winzor, D. J. J. Polym. Sci., Polym. Chem. Ed. 1977,15, 2919) for a polystyrene sample with Afw(0)/Mn(0) = 1.03 and G(S)/G(X) = 1

Quantitative characterization of radiation degradation in polymers by evaluation of scission and cross-linking yields

This article presents a survey of literature on the quantitative characterization of the radiation degradation of polymers in terms of scission and cross-linking. An outline of the theoretical expressions for such characterization is followed by a general discussion of ways in which those equations may be used to quantify radiation chemical yields of scission, G(S), and cross-linking, G(X). Specific considerations of their practical application begin with the classical Charlesby-Pinner treatment of solubility measurements on extensively degraded polymers, and then switch to characterization by means of solution studies of polymer samples subjected to much lower radiation doses. Included in those discussions is the evaluation of G(S) and G(X) by quantifying the effect of irradiation on molecular size distributions inferred from sedimentation velocity, gel permeation chromatography and dynamic light scattering measurements, and also their evaluation from the dose dependence of average molecular weights determined by osmometry, classical light scattering, sedimentation equilibrium and gel permeation chromatography. Copyrigh

Substrate as a source of thermodynamic nonideality in enzyme kinetic studies: Invertase-catalyzed hydrolysis of sucrose

Expressions for the effects of thermodynamic nonideality arising from the use of high concentrations of small substrate in enzyme kinetic studies are derived. Their application to experimental results for the hydrolysis of sucrose by yeast invertase (pH 4.9, 37 °C) signifies that the progressive decrease in initial velocity at high sucrose concentration is consistent with the occurrence of isomeric expansion during the transition of an enzyme-substrate complex to its activated state. Ultracentrifuge studies on the yeast enzyme preparation are then used to establish the physical acceptability of the volume change required to account for the kinetic effects in these terms: the postulated expansion of 1.3 liter/mol would represent a mere 0.16% increase in hydrated volume (or a corresponding increase in extent of asymmetry). Finally, although originally interpreted to signify an effect of sucrose on water concentration, published results for the invertase-sucrose system [J. M. Nelson and M. P. Schubert (1928) J. Amer. Chem. Soc.50, 2188-2193] also find a rational explanation in terms of the present analysis based on effects of thermodynamic nonideality in enzyme kinetic studies

Use of a resonant mirror biosensor to characterize the interaction of carboxypeptidase a with an elicited monoclonal antibody

The binding of apocarboxypeptidase A to an immobilized form of its elicited monoclonal antibody has been used to explore the potential of a biosensor instrument (IAsys) based on resonant mirror technology for the quantitative characterization of antibody-antigen interactions. Advantage has been taken of the stirred cuvette design of the IAsys instrument to develop a stepwise titration procedure for thermodynamic characterization of the interaction, an association equilibrium constant of 3.3 (±0.9) x 10 M having been obtained under the conditions studied (0.1 M Tris/HCl-0.5 M NaCl, pH 7.5, 21°C). In a test of the feasibility of subjecting the time course of biosensor response to conventional pseudo-first-order kinetic analysis, nonconformity of results with such description was encountered at high and low concentrations of apocarboxypeptidase A. Whereas the deviations from Langmuirian kinetic behavior at high antigen concentrations undoubtedly stem from the same sources as those already encountered in studies with the BIA- core biosensor instrument, the deviations at the other concentration extreme occur in a range in which the assumed constancy of free antigen concentration in the liquid phase is becoming a poor approximation. An alternative approach in such circumstances has been tested in which prior thermodynamic characterization is a prerequisite for rate constant evaluation by means of a second-order kinetic analysis. Finally, the effect of soluble anticarboxypeptidase A on the pseudo-first-order kinetics of the biosensor response has been used to illustrate a simple kinetic procedure for evaluating the affinity constant for the antibody-antigen interaction in solution, a value of 1.9 (±0.2) x 10 M being obtained by such means

Thermodynamic nonideality in macromolecular solutions. Evaluation of parameters for the prediction of covolume effects.

Second virial coefficients and hence covolumes for self‐interaction of five proteins, viz. ribonuclease, ovalbumin, bovine serum albumin, catalase and α‐crystallin, have been determined by analyzing the concentration dependence of the partition coefficient obtained from frontal chromatographic studies on either Fractogel TSK HW55 or porous glass beads. The resulting estimates of the effective radii essentially duplicate their Stokes counterparts and thereby provide further justification for assuming the approximate identity of the thermodynamic and hydrodynamic radii of hydrated globular proteins. Gel chromatographic evaluation of second virial coefficients for protein/dextran systems has led to elimination of the sphere/sphere model as a valid thermodynamic description of the space‐filling effects in protein/polymer mixtures, since it does not predict the observed independence of covolume, expressed per unit mass of polymer, upon size of the polymer. This requirement is met by the sphere/rod model [Edmond, E. & Ogston, A. G. (1968) Biochem. J. 109, 569–576] and also by the sphere/flexiblesegment model [Hermans, J. (1982) J. Chem. Phys. 77, 2193–2203]. Furthermore, similar studies of the effect of solute radius on covolume for interaction with dextran T70 attest to the adequacy of either model for predicting the thermodynamic nonideality arising from the inclusion of dextrans in protein solutions, and also provide the relevant calibration of the model