Nonequivalence of second virial coefficients from sedimentation equilibrium and static light scattering studies of protein solutions Original Research Article

Experimental data for ovalbumin and lysozyme are presented to highlight the nonequivalence of second virial coefficients obtained for proteins by sedimentation equilibrium and light scattering. Theoretical considerations confirm that the quantity deduced from sedimentation equilibrium distributions is B22, the osmotic second virial coefficient describing thermodynamic nonideality arising solely from protein self-interaction. On the other hand, the virial coefficient determined by light scattering is shown to reflect the combined contributions of protein–protein and protein–buffer interactions to thermodynamic nonideality of the protein solution. Misidentification of the light scattering parameter as B22 accounts for published reports of negative osmotic second virial coefficients as indicators of conditions conducive to protein crystal growth. Finally, textbook assertions about the equivalence of second virial coefficients obtained by sedimentation equilibrium and light scattering reflect the restriction of consideration to single-solute systems. Although sedimentation equilibrium distributions for buffered protein solutions are, indeed, amenable to interpretation in such terms, the same situation does not apply to light scattering measurements because buffer constituents cannot be regarded as part of the solvent: instead they must be treated as non-scattering cosolutes.