Reexamining protein–protein and protein–solvent interactions from Kirkwood-Buff analysis of light scattering in multi-component solutions

Abstract

The classic analysis of Rayleigh light scattering (LS) is re-examined for multi-component protein solutions, within the context of Kirkwood-Buff (KB) theory as well as a more generalized canonical treatment. Significant differences arise when traditional treatments that approximate constant pressure and neglect concentration fluctuations in one or more (co)solvent/co-solute species are compared with more rigorous treatments at constant volume and with all species free to fluctuate. For dilute solutions, it is shown that LS can be used to rigorously and unambiguously obtain values for the osmotic second virial coefficient (B 22 ), in contrast with recent arguments regarding protein interactions deduced from LS experiments. For more concentrated solutions, it is shown that conventional analysis over(under)-estimates the magnitude of B 22 for significantly repulsive(attractive) conditions, and that protein-protein KB integrals (G 22 ) are the more relevant quantity obtainable from LS. Published data for α-chymotrypsinogen A and a series of monoclonal antibodies at different pH and salt concentrations are re-analyzed using traditional and new treatments. The results illustrate that while traditional analysis may be sufficient if one is interested in only the sign of B 22 or G 22 , the quantitative values can be significantly in error. A simple approach is illustrated for determining whether protein concentration (c 2 ) is sufficiently dilute for B 22 to apply, and for correcting B 22 values from traditional LS regression at higher c 2 values. The apparent molecular weight M 2,app obtained from LS is shown to generally not be equal to the true molecular weight, with the differences arising from a combination of protein-solute and protein-cosolute interactions that may, in principle, also be determined from LS.

Rigorous analysis of static light scattering measurements on buffered protein solutions

“…solutions of ovalbumin [10], chymotrypsinogen A [11], and IgG1 [23,24]. 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 ordinate intercept of a Debye plot for protein solutions is predicted to underestimate M 2 , this potential deficiency of static light scattering for the measurement of protein molecular mass in aqueous solution has only resurfaced recently [10,11,23]. Ordinate intercepts signifying A 1 values greater than unity have been reported for buffered aqueous…”

“…The anomaly has been traced to disregard of the role of buffer and supporting electrolyte components, as if these cosolutes have no effect on the overall intensity of light scattering, even though such effects were described more than fifty years ago [7.8]. Reconsideration of how cosolutes affect the way macromolecules scatter light [9][10][11] has explained the negative light scattering second virial coefficients [2][3][4], reflecting situations in which the B 22 contribution is outweighed by an opposing protein-cosolute counterpart at high cosolute concentration.…”

Concentration dependence of translational diffusion coefficients for globular proteins

“…Although concentration dependence of the translational diffusion coefficient for macromolecular solutes has received considerable theoretical scrutiny, much of that attention has been directed towards the description of diffusion in quasielastic lights scattering spectroscopy (dynamic light scattering) studies, 6,[11][12][13][14][15][16] where the solute chemical potential is being monitored under the constraint of either constant pressure [17][18][19] or constant colume: 20 either option applies to the incompressible solutions being considered here. As noted by Phillies, 15 the disparate theoretical description of D-c dependence reported by Batchelor 4 refers to the situation involving ux of solute molecules under the inuence of an applied force, the gradient in solute chemical potential under the constraint of constant solvent chemical potential -a constraint that allows all buffer and small electrolyte components to be regarded as part of the solvent.…”

Characterization of Protein-Protein Interactions via Static and Dynamic Light Scattering

“…We will cite from these without further reference. More rigorous publications, particularly those dealing with non-ideal, multi-component solutions are found in the scientific literature (e.g., Blanco et al, 2011, and references therein).…”