This thesis involves the examination and description of the effects of thermodynamic non-ideality arising from the space-filling properties of solute species present in the system. Both developmental and applied aspects of nonideality are investigated.Expressions are derived in Chapter 1 for the effects of thermodynamic non-ideality arising from the use of high concentrations of small substrate in enzyme kinetic studies. 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 concentrations is consistent with the occurence of isomeric expansion during the transition of 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 litre/mol would represent a mere 0.16% increase in hydrated volume (or a corresponding increase in extent of asymmetry). In addition, although originally interpreted to signify an effect of sucrose on water concentration, published results for the invertase-sucrose system all find rational explanation in terms of the present analysis based on effects of thermodynamic non-ideality in enzyme kinetic studies.In Chapter 2 the space-filling effects of sucrose on the dimerization of α-chymotrypsin have been investigated by sedimentation equilibrium studies of the enzyme in acetate-chloride buffer, pH 3.9, I 0.2. From the extent of enhancement of the apparent dimerization constant in the presence of 0.05 - 0.16 M sucrose it is concluded that this effect of thermodynamic non-ideality finds quantitative explanation in terms of excluded volume. Results of sedimentation equilibrium experiments on α-chymotrypsin in the presence of 0.1 M glycerol were also shown to be consistent with interpretation in terms of the model of space-filling effects entailing complete exclusion of small solute from the hydrated protein domain. However, the suggested approximation that the radius of small solute would be sufficiently small to be neglected in the calculation of covolumes [Winzor D.J. and Wills P.R., (1986) Biophys. Chem. 25, 243-251 DOI:10.1016/0301-4622(86)80016-3] has not withstood the more stringent test afforded by the present study of α-chymotrypsin dimerization: an effective thermodynamic radius for sucrose of 0.34 nm was employed in those calculations, a value inferred from the covolume for self-interaction (αM,M) obtained by frontal gel chromatography on Sephadex G-10 under the conditions of the ultracentrifugal studies. Finally, the utility of liquid-liquid partitioning as an alternative means of assessing activity coefficients of small solutes (αS,M) has been demonstrated.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 in Chapter 3 by analysing 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 g. of polymer, upon size of the polymer. This requirement is met by the sphere-rod model [Edmond, E. and Ogston, A.G. (1969) Biochem. J. 109, 569-576] and also by the sphere-flexible segment model [Hermans, J. (1982) J. Chem. Phys. 77, 2193-2203 DOI:10.1063/1.444026]. 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 non-ideality arising from the inclusion of Dextrans in protein solutions, and also provide the relevant calibration of the model.A combination of ultrafiltration with either equilibrium dialysis or frontal gel chromatography has been used to evaluate the effects of thermodynamic non-ideality in mixtures of bovine serum albumin and charged ligands in Chapter 4. Studies with methyl orange, chlorpromazine and chromate as ligand all demonstrated inadequacy of the Donnan effect for description of the difference between the concentrations of free ligand in a mixture and the protein-free phase with which it is in dialysis equilibrium. On the basis of a quantitative relationship derived for the situation in which Donnan and thermodynamic non-ideality effects both operate, values of the second virial coefficient for albumin and ligand have been determined. For albumin and either methyl orange or chlorpromazine the magnitude of this second virial coefficient has been rationalized on the statistical mechanical basis of excluded volume. For the albumin-chromate system, however, the thermodynamic non-ideality was manifested as a negative deviation from Raoult's Law, in keeping with the classical behaviour of electrolyte ions. From the viewpoint of the characterization of ligand binding, a unique feature of the ultrafiltration-gel chromatography and ultrafiltration-equilibrium dialysis methods is their ability to define not only the binding function but also the activity coefficient of ligand for a given acceptor-ligand mixture. Consequently, irrespective of whether the ligand is charged or uncharged, the intrinsic binding constant that is determined is the thermodynamic parameter instead of the apparent value that is obtained from methods based on assumed thermodynamic non-ideality.The thesis concludes with the application of thermodynamic non-ideality as a probe of specific protein interactions. In section A of Chapter 5, results of a sedimentation equilibrium study of the inhibitory effect of calcium ion on the dimerization of α-chymotrypsin (pH 3.9, I 0.2) are used to establish that the phenomenon does not reflect increased electrostatic repulsion between Ca2+-saturated enzyme molecules, but rather the displacement of the monomer-dimer equilibrium by the specific, weak interaction of metal ion with a single site on a monomeric enzyme.Thermodynamic non-ideality arising from the space-filling effects of added sucrose or glycerol has been employed in section B to confirm that the reversible unfolding of ribonuclease effected by acid and temperature should be considered in equilibrium terms. Whereas high concentrations of these two solutes have no effect on the pH-profile for the acid expansion of bovine serum albumin, the inclusion of sucrose (0.5 M) in ribonuclease solutions elicits displacement of the spectrally determined pH-profile for the unfolding in the acidic direction, a finding that is consistent with the action of sucrose as an inert space-filling solute on an isomerization equilibrium between native and acid-expanded states of the enzyme. The ratio of apparent isomerization constants, at any given pH, in the presence and absence of sucrose was therefore interpreted on the statistical mechanical basis of excluded volume to determine the change in enzyme volume required for the displacement to reflect thermodynamic non-ideality. Since the extent of the volume increase (5%) that is calculated by this means matches the value from viscosity measurements, the effect of sucrose on the unfolding of ribonuclease is entirely attributable to thermodynamic non-ideality arising from the space-filling property of this small solute. On the other hand, quantitative reappraisal of published results on the effects of glycerol on the thermal denaturation of ribonuclease at pH 2.8 [Gekko, K. and Timasheff, S.N. (1981) Biochemistry 20, 4677-4686 DOI:10.1021/bi00519a024] leads to an estimated volume increase that is much smaller than that inferred from hydrodynamic studies, a disparity attributed to the dual actions of glycerol as a space-filling solute and as a ligand that binds preferentially to the thermally unfolded form of the enzyme. Although the effects of glycerol on melting curves for ribonuclease are not interpretable solely on the basis of excluded volume, this deficiency of the quantitative analysis does not detract from the observation that glycerol exerted a net excluded volume effect, and hence the conclusion that the unfolding transition is an equilibrium phenomenon. These findings suffice to illustrate the utility of thermodynamic non-ideality as a probe for ascertaining the validity of considering an interconversion between native and unfolded states in terms of an equilibrium mechanism, which has been an inherent but untested assumption in most studies of protein denaturation.