ANALYSES OF THERMODYNAMIC DATA FOR CONCENTRATED HEMOGLOBIN-SOLUTIONS USING SCALED PARTICLE THEORY - IMPLICATIONS FOR A SIMPLE 2-STATE MODELOF WATER IN THERMODYNAMIC ANALYSES OF CROWDING IN-VITRO AND IN-VIVO

Quantitative description of the thermodynamic consequences of macromol ecular crowding (excluded volume nonideality) is an important componen t of analyses of the thermodynamics and kinetics of noncovalent intera ctions of biopolymers in vivo and in concentrated polymer solutions in vitro. By analyzing previously published thermodynamic data, we have investigated extensively the comparative applicability of two forms of scaled particle theory (SPT). In both forms, macromolecules are treat ed as hard spheres, but MSPT, introduced by Ross and Minton, treats th e solvent as a structureless continuum, whereas bulk water molecules a re included explicitly as hard spheres in BSPT, an approach developed by Berg. Here we use both MSPT and BSPT to calculate the excluded volu me component of the macromolecular activity coefficient of hemoglobin (Hb) at concentrations up to 509 mg/ml by fitting osmotic pressure dat a for Hb and sedimentation equilibrium data for Hb and sickle-cell Hb (HbS). Both forms of SPT also are used here to analyze the effects of other globular proteins (BSA and Hb) on the solubility of HbS. In appl ying MSPT and BSPT to analyze macromolecular crowding, the extent of h ydration delta(Hb) (in gH(2)O/gprotein) is introduced as an adjustable parameter to specify the effective (hard sphere) radius of hydrated H b. In our nonlinear least-squares fittings based on BSPT, the hard sph ere radius of bulk water molecules is either fixed at 1.375 Angstrom o r floated. Although both forms of SPT yield good fittings (with differ ent values of delta(Hb)) at Hb concentrations up to 350 mg/ml only BSP T gives good fittings of all available Hb osmotic pressure data as wel l as of the sedimentation equilibrium and solubility data. Only BSPT p redicts values for delta(Hb) (similar to 0.5-0.6 g/g) in the range obt ained for Hb from hydrodynamic measurements (similar to 0.36-0.78 g/g) . These findings indicate the applicability, at least in the context o f BSPT, of a simple two-state classification of water (bulk water and water of macromolecular hydration) as a basis for interpreting exclude d volume nonideality in concentrated solutions of globular proteins.