Biomedical subjects

D J Winzor

Publications and source records attributed to D J Winzor.

At least 55 records · Page 3Linked to original sources

PubMed1996

Interpretation of deviations from pseudo-first-order kinetic behavior in the characterization of ligand binding by biosensor technology.

Macromolecular interactions observed using surface plasmon resonance technology (BIAcore, Pharmacia) often display kinetic behavior which deviates from the pseudo-first-order time dependence that has been predicted for 1:1 interactions of ligand and ligate. In the present study we reviewed the majors reasons for such deviations, and present results which suggest that the most common source of deviations from the pseudo-first-order kinetic approximation of BIAcore kinetic data is likely to be heterogeneity of the immobilized ligand sites. A simplified analysis of the adsorption stage of BIAcore data is presented in terms of the net observed pseudo-first-order rate constant, kobs, rather than in terms of the association and dissociation rate constants, ka and kd. The analysis is then extended to the determination of the dissociation equilibrium constant for the interaction of ligand and ligate in the solution phase from sensorgrams reflecting competition between soluble and immobilized forms of ligand for ligate.

Adsorption↗

PubMed1996

Demonstration of an upper limit to the range of association rate constants amenable to study by biosensor technology based on surface plasmon resonance.

Numerical simulation of BIAcore sensorgrams has highlighted the need for concern about an assumption, inherent in current determinations of rate constants for macromolecular interactions, that the concentration of solute in the flowing phase remains constant at its injected value. This assumption is shown to be valid for systems with effective association rate constants equal to or less than 10 M(-1) values characteristic of antibody interactions with protein antigens. However, the assumption loses validity when the effective association rate constant is raised to 10 M' . The basic correctness of the latter prediction is verified by an experimental study of the interaction between soybean trypsin inhibitor and immobilized -trypsin, a system with comparable reaction kinetics.

Animals↗

PubMed1995

Effects of solute multivalence on the evaluation of binding constants by biosensor technology: studies with concanavalin A and interleukin-6 as partitioning proteins.

The interaction of concanavalin A with immobilized carboxylmethyldextran has been characterized by means of a biosensor based on surface plasmon resonance detection. Adsorption and desorption of this bivalent lectin to/from the biosensor surface are shown to deviate markedly from pseudo-first-order kinetics, an assumption inherent in the usual kinetic approach to the characterization of interactions by biosensor technology. Similar results for the interaction of a dimeric and hence bivalent form of human interleukin-6 with its receptor immobilized on the biosensor plate support the conclusion that this deviation from pseudo-first-order kinetics originates from multivalence of the partitioning protein. Use of the kinetic approach to characterize the binding of multivalent proteins to immobilized affinity sites on the biosensor chip is therefore precluded because of nonconformity with the model on which the quantitative analysis is based. Instead, an intrinsic binding constant of 2.5 x 10(5) M-1 for the interaction of concanavalin A with the carboxymethylated dextran layer coating the biosensor chip has been obtained by interpreting the equilibrium biosensor responses in terms of expressions developed in the context of quantitative affinity chromatography of multivalent partitioning solutes.

Adsorption↗

PubMed1995

Use of a biosensor with surface plasmon resonance detection for the determination of binding constants: measurement of interleukin-6 binding to the soluble interleukin-6 receptor.

The interaction of recombinant human interleukin-6 (IL-6) with the soluble extracellular form of its receptor (sIL-6R) has been characterized by the application of expressions developed for quantitative affinity chromatography to results obtained with a biosensor based on surface plasmon resonance detection. First, the interaction of sIL-6R with IL-6 covalently attached to the biosensor-chip was characterized from the dependence of the surface plasmon resonance response upon the concentration of receptor injected into the biosensor. A binding constant for the interaction between sIL-6R and IL-6 was then determined from the biosensor response observed for mixtures of IL-6 and receptor--a procedure that is shown to provide unequivocal characterization of the competing reaction, irrespective of the model used to describe the biphasic interaction between partitioning receptor and immobilized IL-6. A binding constant of 5 x 10(7) M-1 has been obtained for the interaction of sIL-6R with two equivalent and independent sites on an essentially dimeric IL-6 preparation produced using the pUC vector system, and also for the interaction of sIL-6R with a monomeric IL-6 preparation that was univalent in its interaction with receptor.

Amino Acid Sequence↗

PubMed1995

The rectangular hyperbolic binding equation for multivalent ligands.

An improved procedure for the characterization of interactions involving multivalent ligands which eliminates a previous requirement that interaction parameters for independent and equivalent binding be evaluated from a general counterpart of the Scatchard analysis is described. Binding data for the interaction of aldolase with muscle myofibrils are then used to illustrate application of the procedure, which allows the stoichiometry and intrinsic binding constant to be evaluated by standard nonlinear regression analysis in terms of a rectangular hyperbolic relationship.

Fructose-Bisphosphate Aldolase↗

PubMed1995

Characterization of the interactions of NADH with the dimeric and tetrameric states of methaemoglobin.

The binding of NADH to the dimeric (alpha beta) and tetrameric (alpha 2 beta 2) states of human aquomethaemoglobin has been characterized by sedimentation equilibrium studies of the effect of the concentration of free ligand on the macromolecular state of the haemoprotein. Both macromolecular states of aquomethaemoglobin exhibit a single binding site for NADH, which interacts approximately tenfold more strongly (6000 cf. 700 M-1) with the tetramer under the conditions studied (pH 6.0, I 0.10). Because the structure of aquomethaemoglobin resembles that of the deoxy state of haemoglobin, there is a high probability that organic phosphates also bind to dimeric deoxyhaemoglobin, a phenomenon which is not considered in thermodynamic treatments of the interplay between oxygen binding and its allosteric inhibition by 2,3-bisphosphoglycerate. Fortunately, the equilibrium constant for deoxyhaemoglobin self-association is so large that neglect of the interaction between allosteric inhibitor and dimeric haemoglobin is an oversight that should have no deleterious implications in the resultant thermodynamic analysis of the interplay between the preferential interactions of oxygen and organic phosphate with the various macromolecular states of deoxyhaemoglobin.

Binding Sites↗

PubMed1995

Effects of heterogeneity and cooperativity on the forms of binding curves for multivalent ligands.

An exploratory investigation is made of the binding behavior that is likely to be encountered with multivalent ligands under circumstances where a single intrinsic binding constant does not suffice to describe all acceptor-ligand interactions. Numerical simulations of theoretical binding behavior have established that current criteria for recognizing heterogeneity and cooperativity of acceptor sites on the basis of the deviation of the binding curve from rectangular hyperbolic form for univalent ligands also apply to the interpretation of the corresponding binding curves for multivalent ligands. However, for systems in which the source of the departure from equivalence and independence of binding sites resides in the ligand, these criteria are reversed. On the basis of these observations a case is then made for attributing results of an experimental binding study of the interaction between pyruvate kinase and muscle myofibrils to positive cooperativity of enzyme sites rather than to heterogeneity or negative cooperativity of the myofibrillar sites.

Kinetics↗

PubMed1995

Measurement of thermodynamic nonideality arising from volume-exclusion interactions between proteins and polymers.

The effective thermodynamic radii of 23 ribosomal proteins from the 50 S subunit have been determined by gel chromatography on Sephadex G-50, thereby supporting the contention that most of the proteins of the 50 S ribosomal unit exhibit reasonably globular structures. To investigate further the usefulness of modelling proteins as spheres, the second virial coefficient describing excluded volume interactions of some ribosomal proteins with two inert polymers, polyethylene glycol (PEG) and dextran, has been determined by gel chromatography and/or sedimentation equilibrium techniques. Protein-polymer excluded volumes obtained with PEG 20000 and Dextran T70 as the space-filling solute are shown to conform reasonably well with a quantitative expression describing interaction between an impenetrable sphere and an ideal Brownian path (K.M. Jansons and C.G. Phillips, J. Colloid Interface Sci., 137 (1990) 75).

Journal Article↗

PubMed1995

Stabilizing effect of sucrose against irreversible denaturation of rabbit muscle lactate dehydrogenase.

Measurements of the kinetics of activity loss by rabbit muscle lactate dehydrogenase in acetate-chloride buffer, pH 5.0, I 0.20, have shown that the enzyme exhibits greater stability against irreversible inactivation when the buffer is supplemented with sucrose (0.1 M-0.5 M). On the basis of sedimentation equilibrium distributions obtained for enzyme in the absence and presence of sucrose (0.5 M), the lactate dehydrogenase is essentially dimeric in both environments. The observed stabilization of enzyme activity has therefore been considered in terms of the space-filling effects of sucrose on an isomerization equilibrium between native and unfolded forms of dimeric lactate dehydrogenase, which precedes irreversible inactivation of the unfolded isomer. Interpretation of the kinetic results on that basis has led to the conclusion that the initial stage of enzyme unfolding entails a minor change in volume and/or asymmetry of the lactate dehydrogenase that gives rise to a 4% increase in the second virial coefficient describing excluded volume interactions between dimeric enzyme and sucrose.

Animals↗

PubMed1995

Thermodynamic nonideality of enzyme solutions supplemented with inert solutes: yeast hexokinase revisited.

Published experimental results on the activating effect of polyethylene glycol on the interaction of yeast hexokinase with glucose (R.P. Rand, N.L. Fuller, P. Butko, G. Francis and P. Nicholls, Biochemistry, 32 (1993) 5925) are reinterpreted in statistical-mechanical terms of excluded volume. Of particular interest is the ability of this standard treatment of thermodynamic nonideality to accommodate the observed non-exponential dependence of the activation upon osmotic pressure of the polyethylene glycol solution--a dependence which is not predicted by analyses based on the concept of osmotic stress that was invoked originally to account for the results.

Enzyme Activation↗

PubMed1994

Elimination of self-association as the source of the thermodynamic nonideality in aqueous proline solutions.

The effect of high concentrations of proline on the diffusion coefficient of water has been examined to assess the extent to which the resulting thermodynamic nonideality could be explained on the statistical-mechanical basis of excluded volume. In fact, such a space-filling role not only accounts for the proline concentration-dependence of the diffusion coefficient of water but it also accounts for the nonideality of proline in freezing point depression and isopiestic measurements. These findings refute the conclusion (Schobert, B. and Tschesche, H. (1978) Biochim. Biophys. Acta 541, 270-277) that the stabilization of enzyme structure by high concentrations of proline stems from self-association of the imino acid via intermolecular hydrogen bonding; and thereby support the concept that the protective effect of proline on enzyme stability must reside mainly in its action as an inert, space-filling solute.

Diffusion↗

PubMed1994

Effects of molecular crowding on protein self-association: a potential source of error in sedimentation coefficients obtained by zonal ultracentrifugation in a sucrose gradient.

Theoretical and experimental studies have illustrated a potential source of error in sedimentation coefficients obtained by sucrose density gradient centrifugation of proteins undergoing reversible self-association. The error stems from the excluded volume (molecular crowding) effect of the sucrose on the activity coefficients of monomeric and polymeric states. The consequent displacement of the equilibrium position in favor of polymeric state(s) is a function of sucrose concentration, and can therefore result in failure to detect the equilibrium coexistence of monomer if 5% sucrose suffices to displace the equilibrium completely toward dimer. In less extreme situations, it may result in the evaluation of an average sedimentation coefficient whose magnitude is a function of sucrose concentration and hence of the distance migrated into the sucrose gradient. These features are illustrated by the results of computer-simulated sedimentation of reversibly dimerizing systems in a sucrose gradient, and by conventional sedimentation velocity experiments on yeast enolase.

Centrifugation, Zonal↗

PubMed1993

The problem of partial competition in the quantitative characterization of interactions by competitive binding assays.

Binding expressions are derived and analytical procedures developed for the quantitative characterization of inhibitor binding that is only partially competitive with the interaction between an acceptor and the ligand that is being monitored. Two such situations are considered: (i) that in which the partial competition reflects binding of inhibitor to fewer acceptor sites than available to ligand; and (ii) that in which the partial competition reflects binding of inhibitor to acceptor sites in addition to those occupiable by ligand. The potential efficacy of the suggested analyses is then explored by their application to simulated data that span the likely range of experimental behavior. Quantitative analysis of the displacement of [3H]nitrobenzylthioinosine from cultured leukemic cells by an adduct of 5'-S-(2-amino-ethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine with fluorescein-5-isothiocyanate is used to establish that the cells possess 6% fewer sites (150,000 cf. 159,000 sites/cell) for the fluorescent adduct than for the tritiated ligand, and that the binding is 10-fold weaker (binding constant of 0.28 cf. 2.8 nM-1). Corresponding analysis of results obtained with bovine aorta endothelial cells indicates that a 3-fold weaker interaction (binding constant of 1.1 cf. 3.3 nM-1) occurs between the fluorescent adduct and 79% of the cell sites accessible to the tritiated ligand. The present analytical procedures extend the utility of competitive binding assays for the quantitative screening of potential inhibitors by removing the inherent limitation of existing analyses that all acceptor sites be accessible to both the competing solute and the indicator ligand.

Adenosine↗

PubMed1993

Thermodynamic nonideality in macromolecular solutions: interpretation of virial coefficients.

Consideration is given to the interpretation of virial coefficients reflecting thermodynamic nonideality in incompressible solutions of a single macromolecular species for which there is no volume change on mixing. Expressions are presented for the concentration dependence of thermodynamic activity under conditions where either the chemical potential of solvent or the pressure is fixed, these two conditions being mutually exclusive. For the former situation, which applies to partition equilibrium procedures, the thermodynamic activity is most conveniently defined on the molar scale because the coefficients in polynomial expansions for the osmotic pressure and the activity coefficient in terms of molarity are then related to each other without the inclusion of partial molar volume terms. Under conditions of constant pressure a similar situation prevails provided that the osmotic pressure and the corresponding activity coefficient are expanded in powers of molality. In either case conversion of the virial expansions to the other concentration scale is possible, but requires the introduction of partial molar volume terms into the virial coefficients. The implications of these findings are discussed in relation to results obtained by osmometry, isopiestic measurements, equilibrium dialysis, gel chromatography, and sedimentation equilibrium.

Chromatography, Gel↗

PubMed1992

The influence of pH on the interaction of lipophilic anthracyclines with bovine serum albumin. Quantitative characterization by measurement of fluorescence quenching.

We have investigated the interaction of the lipophilic anthracyclines 4'-iodo-4'-deoxydoxorubicin (IDX) and 4-demethoxy-daunorubicin (DDN) with bovine serum albumin by the quantitation of fluorescence quenching. The protein binding of IDX was extremely sensitive to the pH of the solution in which the complex was formed and paralleled the effect of pH on dimerization of the drug. The effect of pH on the protein binding and self-association of DDN was less extensive. Both compounds exhibited curvilinear Scatchard plots indicating apparent cooperativity in the binding process. Because of the self-association of the drugs in aqueous solution, we attempted to resolve this cooperativity in terms of the preferential binding of the dimer to the acceptor. However, we found that similar Scatchard plots could be simulated by using slightly erroneous estimates of the fluorescence yield of the complex, rendering any such analysis inconclusive. Consequently, the relationship between acceptor concentration and the fraction of ligand bound was considered to be fitted adequately in terms of a single acceptor site per albumin molecule. The pH dependence of the association constants for bovine serum albumin was described best by the hydrophobic interaction of neutral drug monomer with a binding site with titratable affinity. We postulate that the pH-dependent binding of some anthracyclines with albumin may lead to their enhanced uptake, relative to that of non-target organs, into tumours with an acidotic extracellular milieu.

Doxorubicin↗

PubMed1992

Study of high-affinity interactions by quantitative affinity chromatography. Analytical expressions in terms of total ligand concentration.

Analytical expressions are derived for the description of ligand-facilitated and ligand-retarded desorption of partitioning solute in terms of total ligand concentration in quantitative affinity chromatography. Their application is then illustrated by consideration of results from recycling partition equilibrium studies of the heparin-facilitated desorption of thrombin from heparin-Sepharose, and of the competition between methyl-alpha-D-mannoside and p-nitrophenyl-alpha-D- mannoside for concanavalin A immobilized on CPG-170. Finally, published frontal affinity chromatographic data for the NADH-dependent elution of rabbit muscle lactate dehydrogenase from oxamate-Sepharose are reanalysed using these equations to demonstrate the characterization of a system reflecting the binding of a solute-ligand complex to an affinity matrix. This investigation extends the scope of quantitative affinity chromatography to include not only the study of solute-ligand interactions governed by larger binding constants but also the characterization of interactions in which the partitioning solute and ligand are both macromolecular, and eliminates the need for prior dialysis to establish the free ligand concentration required for application of earlier analytical expressions.

Animals↗

PubMed1992

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.

Animals↗

PubMed1992

Rationalization of the effects of compatible solutes on protein stability in terms of thermodynamic nonideality.

Inhibition by compatible solutes such as proline and glycine betaine of the rate of coagulation, at 60 degrees C, of bovine serum albumin in 0.1 M acetate buffer, pH 5, is used as a model system to substantiate the concept that the production of high concentrations of osmolytes by plants and other organisms in response to stress (e.g., drought) results in stabilization of native enzyme structures via nonspecific excluded volume effects. The paradoxical situation whereby this effect of compatible solutes counters to some extent the protein-precipitating effect of poly(ethylene glycol) is also seemingly resolved.

Betaine↗