This thesis commences with two Chapters concerned with methodological aspects of the study of ligand binding to macromolecular acceptors. First, a slightly modified but theoretically analogous version of the rate-of-dialysis method for investigating ligand binding [Colowick, S.P. and Womack, F.C. (1969) J. Biol. Chem. 244, 774-777] has been developed in order to examine more closely the principle of the procedure. It is shown that the method is described by the theory that pertains to measurement of diffusion coefficients by the diaphragm cell method. Secondly, a recycling gel partition technique has been developed for acceptor-ligand systems that exhibit isosbestic points, its use being illustrated with a study of the binding of methyl orange to bovine serum albumin. A combination of binding studies and sedimentation velocity experiments is then used to dispel the concept [Klotz, I.M. and Urquhart, J.M. (1949) J. Phys. Colloid Chem. 53, 100-114 DOI: 10.1021/j150466a008 ] that the binding curve for this system shows a dependence upon the albumin concentration used for its measurement.
In Chapter 3 a technique for the measurement of the net charge of a protein is reported which depends upon assessment of the Donnan effect by a combination of equilibrium dialysis and ultrafiltration in conjunction with conductivity measurements. Studies with ovalbumin, bovine serum albumin and lysozyme are used to illustrate the method, a decided advantage of which is the absence of assumptions about the extent of specific ion binding.
Advantage is then taken, in Chapter 4, of this procedure for valence estimation to examine the concept of charge conservation in two macromolecular interactions. First, concomitant measurement of the binding function and the net charge for a series of bovine serum albumin-methyl orange mixtures (pH 7.4, I = 0.05 M) showed that the magnitude of the negative charge on the albumin increased linearly with the number of molecules of methyl orange bound to the protein, the observed slope (0.96 ± 0.08) being in excellent agreement with that predicted on the basis of charge conservation for attachment of this univalent, negatively charged ligand. Secondly, in a study of the self-associating enzyme α-chymotrypsin at pH 3.9, I = 0.11 M, the net charge (expressed per base-mole) was shown to be invariant (+10) in solutions in which the mole fraction of monomeric enzyme varied between 0.47 and 0.88, the extent of the range having been established by means of sedimentation equilibrium studies of this monomer-dimer system.
Chapter 5 considers the effects of thermodynamic nonideality on the quantitative characterization of the interaction between a small ligand and a macromolecular acceptor by two types of experimental procedure. The first involves determination of the concentration of ligand in dialysis equilibrium with acceptor-ligand mixture; and the second, direct measurement of the concentration of unbound ligand in the reaction mixture. Explicit expressions are formulated for the appropriate binding functions, with allowances made for composition-dependent nonideality effects assessed on the statistical mechanical basis of covolumes; and then applied to experimental studies on the binding of L-tryptophan and of methyl orange to bovine serum albumin. By showing that the theoretically predicted dependence of the binding functions on acceptor concentration is likely to be insignificant experimentally, this study provides reassurance that the consequences of thermodynamic nonideality in the multiple binding of ligands to a single macromolecular state of a protein are likely to be second-order effects.
This thesis concludes with consideration of the possibility that thermodynamic nonideality arising from inclusion of an inert macromolecular solute may provide the means for detecting isomeric equilibria. Gel chromatographic results obtained in the presence and absence of Dextran T70 and T10 show that the acid-expansion of bovine serum albumin should not be considered in terms of a pH-dependent isomerization equilibrium between native and acid-expanded states, a conclusion confirmed by examining the effect of rotor speed on the sedimentation coefficient of albumin at pH 3.2. Theoretical considerations of the aspartate transcarbamoylase system have indicated that differential gel chromatographic studies in the presence and absence of Dextran T70 have the potential to prove the existence of the isomerization equilibrium (ligand-induced or pre-existing) responsible for the allosteric behaviour of this enzyme. It would appear that nonideality may provide the means of distinction, for some systems at least, between the Monod-Wyman-Changeux and Koshland-Némethy-Filmer models of allostery, which hitherto have been conceptually different but thermodynamically indistinguishable.