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

Quantitative description of the thermodynamic consequences of macromolecular crowding (excluded volume nonideality) is an important component of analyses of the thermodynamics and kinetics of noncovalent interactions 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 treated as hard spheres, but MSPT, introduced by Ross and Minton, treats the solvent as a structureless continuum, whereas bulk water molecules are included explicitly as hard spheres in BSPT, an approach developed by Berg. Here we use both MSPT and BSPT to calculate the excluded volume component of the macromolecular activity coefficient of hemoglobin (Hb) at concentrations up to 509 mg/ml by fitting osmotic pressure data 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 applying MSPT and BSPT to analyze macromolecular crowding, the extent of hydration delta(Hb) (in gH(2)O/gprotein) is introduced as an adjustable parameter to specify the effective (hard sphere) radius of hydrated Hb. In our nonlinear least-squares fittings based on BSPT, the hard sphere radius of bulk water molecules is either fixed at 1.375 Angstrom or floated. Although both forms of SPT yield good fittings (with different values of delta(Hb)) at Hb concentrations up to 350 mg/ml only BSPT gives good fittings of all available Hb osmotic pressure data as well as of the sedimentation equilibrium and solubility data. Only BSPT predicts values for delta(Hb) (similar to 0.5-0.6 g/g) in the range obtained for Hb from hydrodynamic measurements (similar to 0.36-0.78 g/g). These findings indicate the applicability, at least in the context of BSPT, of a simple two-state classification of water (bulk water and water of macromolecular hydration) as a basis for interpreting excluded volume nonideality in concentrated solutions of globular proteins.