Measurement of the second osmotic virial coefficient for protein solutions exhibiting monomer–dimer equilibrium

Abstract

The second osmotic virial coefficient ( B) is a measure of solution nonideality that is useful for predicting conditions favorable for protein crystallization and for inhibition of aggregation. Static light scattering is the technique most commonly used to determine B values, typically using protein concentrations less than 5 mg/mL. During static light scattering experiments at low protein concentrations, frequently the protein is assumed to exist either as a single nonassociating species or as a combination of assembly states independent of protein concentration. In the work described here, we examined the limit for ignoring weak reversible dimerization ( K d ⩾ 1 mM) by comparing B values calculated with and without accounting for self-association. Light scattering effects for equilibrium dimer systems with K d < 20 mM and K d < 1 mM will significantly affect apparent B values measured for 20 and 150-kDa proteins, respectively. To interpret correctly light scattering data for monomer–dimer equilibrium systems, we use an expanded coefficient model to account for separate monomer–monomer ( B 22), monomer–dimer ( B 23), and dimer–dimer ( B 33) interactions.