Larry D. Ward

Effects of thermodynamic nonideality in kinetic studies

Experimental evidence is presented for concentration dependence of the pseudo-first-order rate constant describing the rate of inversion of sucrose by 2 M HCl; and also of the increase in maximal velocity for the catalytic reduction of pyruvate by lactate dehydrogenase that results from addition of the inert macromolecular solutes bovine serum albumin, ovalbumin, and Dextran T70. These somewhat unusual and seemingly diverse observations are examined in terms of a theory formulated on the basis of two equilibrium reactions, the first describing complex formation between two reactants, and the second isomerization of that complex to an activated state prior to product formation. This formulation permits consideration of activity coefficient ratios relevant to the equilibria and the expression of these ratios as power series in total solution composition. Quantitative assessment of the experimental results is made possible in these terms by estimating the magnitudes of the constant coefficients of the virial expansions as excluded volumes. It is concluded that the result observed in the sucrose inversion study finds rational explanation in thermodynamic nonideality factors governing the overall equilibrium between the reactants and the activated complex of sucrose and hydronium ion. For the enzyme-catalyzed reaction the same general equation applies but particular attention is given to the simplified form that is relevant to high substrate concentrations, where, in the absence of inert compounds, the conventional maximal velocity is approached. In this region an increase in velocity observed upon addition of an inert macromolecular component may be considered explicitly in terms of excluded volume effects related to a shape change in the isomerization between enzyme-substrate complex and its activated state.

Quantitative considerations of the consequences of an interplay between ligand binding and reversible adsorption of a macromolecular solute.

Explicit expressions are derived which determine the equilibrium composition of mixtures comprising a multivalent, insoluble matrix, a multivalent, macromolecular solute (acceptor) and a univalent ligand. With three-reactant mixtures of this type a range of combinations of interactions is possible wherein the ligand interacts with either the acceptor or the matrix, in either event perturbing the acceptor-matrix equilibria. Theory encompassing this range of possibilities is written in terms of a single site-binding constant for each type of interaction to account, in general terms, for both multiple binding and crosslinking effects. These explicit thermodynamic relationships are discussed, with the use of reported findings on several biological systems, in two frameworks. First, it is established that the theory is applicable to the quantitative interpretation of affinity chromatography experiments designed to elucidate the thermodynamic interaction parameters governing the various types of interacting system. Second, it is emphasized that the relationships are also relevant to metabolite-induced changes in the subcellular distribution of macromolecular species.

Adaptation of gel electrophoresis for the quantitative study of specific ion binding: Interaction of phosphate with ovalbumin

A gel electrophoretic method is described for the measurement of relatively weak interactions between proteins and charged ligands, its use being illustrated with a study of the binding of phosphate to ovalbumin, not only as the pure solute but also as a component of egg white. An association equilibrium constant of 200 m-1 is assigned to the interaction of a single dibasic phosphate ion with ovalbumin under the conditions (pH 7.4, I 0.15) used to investigate the phenomenon.

Activation of rabbit muscle lactate dehydrogenase by phosphate: active enzyme gel chromatography and enzyme kinetic studies.

Enzyme kinetic studies are presented which demonstrate the activating effect of phosphate on the conversion of pyruvate to lactate by rabbit muscle lactate dehydrogenase. A simple method of active enzyme gel chromatography is used to preclude the possibility that this effect is due to redistribution of enzyme between tetrameric and dissociated states as the result of preferential binding of phosphate to the tetrameric enzymatic form. By analysis of the kinetic results in terms of an ordered two-substrate mechanism, the source of the activation is traced to enhancement of the strength of the enzyme-NADH interaction, primarily because of an increase in the rate constant for the formation of the binary enzyme-coenzyme complex. Preliminary estimates of the relevant equilibrium constants from the kinetic data indicate that the binding of phosphate to rabbit muscle lactate dehydrogenase leads to a two- to fourfold increase in the intrinsic association constant for the interaction between NADH and the enzyme under the conditions (pH 7.4, I = 0.15) used to study the activation phenomenon.

Self-association of sperm whale metmyoglobin

The solution behavior of sperm whale metmyoglobin in 0.15 I phosphate-chloride buffer, pH 7.2, has been examined by sedimentation equilibrium, frontal gel chromatography, and sedimentation velocity. Results obtained from all three studies are shown to be consistent with a self-association model in which dimerization of the myoglobin is governed by an association equilibrium constant of 0.068 liter/g (580 M-1) at 20 degrees C.

Reappraisal of the binding of myosin subfragment 1 to regulated filaments in terms of the steric model of muscle relaxation.

Abstract The sigmoidal form of the binding curves obtained [ L. E. Greene and E. Eisenberg (1980) Proc. Nat. Acad. Sci. USA 77 , 2616–2620 ] for the interaction of myosin subfragment 1 with regulated filaments is reinterpreted and shown to be consistent with a steric blocking model of muscle relaxation in which the interaction of tropomyosin with the myosin-binding sites on the actin filaments is considered to be an equilibrium process rather than an all-or-none phenomenon. Such an interpretation of the results has the advantage that dependence of the binding curve upon filament concentration is predicted, the previous models based on filament isomerization being deficient in this regard.