Effect of Nonadditive Repulsive Intermolecular Interactions on the Light Scattering of Concentrated Protein−Osmolyte Mixtures

Abstract

The static light scattering of three globular proteins, bovine serum albumin, ovalbumin, and ovomucoid, and binary mixtures of each protein and trimethylamine oxide (TMAO) containing between 10 and 70% protein, were measured as a function of total weight per volume concentration up to 100 g/L. The observed dependence of scattering upon concentration may be accounted for quantitatively by an effective hard sphere model incorporating an extension that takes into account the nonadditive nature of the repulsive intermolecular interaction between protein and TMAO.

Rigorous analysis of static light scattering measurements on buffered protein solutions

“…However, results conflicting with the current prediction of protein molecular mass underestimation by static light scattering have also been reported recently for several proteins [13,15,25,26] as evidence for the validity of considering light scattering by a nonassociating protein in buffer to be amenable to interpretation in terms of single-solute theory [14]. Although the consistency of those light scattering measurements with the molecular masses of these well- …”

“…Although the value of A 2 thereby obtained does, of course, provide a phenomenological description of nonideality in a nonassociating protein solution, the objective of those light scattering studies [13][14][15]25,26] was to employ the second virial coefficients for the prediction of protein activity coefficients on the statistical mechanical basis of the potential-of-mean-force (excluded volume) between molecules [5]. For that mechanistic purpose the practice of substituting an incorrect parameter (A 2 ) for B 22 , the osmotic second virial coefficient to which McMillan-Mayer A C C E P T E D M A N U S C R I P T theory [5] applies, has no theoretical justification.…”

“…For that mechanistic purpose the practice of substituting an incorrect parameter (A 2 ) for B 22 , the osmotic second virial coefficient to which McMillan-Mayer A C C E P T E D M A N U S C R I P T theory [5] applies, has no theoretical justification. This invalidation of a basic tenet of the approach to allowance for the effects of thermodynamic nonideality clearly detracts from the recommended use of static light scattering for the rapid quantitative characterization of weak association equilibria requiring study at high protein concentrations [13][14][15]25,36].…”

“…However, interest in nonideality at extremely high protein concentrations in those studies [12,13,25,26] led to the accumulation of much of the data in the concentration region where the middle term in the denominator of Eq.24 could be neglected; and hence to a curve-fitting analysis with minimal input from data in the low concentration region where the consequences of the A 1 factor predominate -see Fig. 1 for results reported for ovalbumin at neutral pH [13].…”

“…Further evidence for the unacceptability of the single-solute treatment of light scattering measurements on buffered protein solutions [12,13,25,26] = 4N A R 2 3 /3]. Clearly, the effective particle volume that emanates from scaled particle theory is not an accurate estimate of B 22 /4 and cannot be relied upon for the evaluation of activity ACCEPTED MANUSCRIPT…”