Automated measurement of the static light scattering of macromolecular solutions over a broad range of concentrations

Abstract

A method and apparatus for automated measurement of the concentration dependence of static light scattering of protein solutions over a broad range of concentration is described. The gradient of protein concentration is created by successive dilutions of an initially concentrated solution contained within the scattering measurement cell, which is maintained at constant total volume. The method is validated by measurement of the concentration dependence of light scattering of bovine serum albumin, ovalbumin, and ovomucoid at concentrations up to 130 g/L. The experimentally obtained concentration dependence of scattering obtained from all three proteins is quantitatively consistent with the assumption that no significant self-association occurs over the measured range of concentration.

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- …”

“…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…”

“…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]. The limiting slope of the dependence of R  /K s upon protein concentration therefore becomes relatively insensitive to the magnitude of A 1 , and hence provides a reasonably accurate estimate of M 2 .…”

“…examines the consequences of this approximation by subjecting reported CG-MALS results [13,15] to closer thermodynamic scrutiny; and a protocol for the correct interpretation of Debye plots is presented.…”

“…The popularity of static light scattering for the characterization of protein interactions has been boosted considerably by the development of an automated procedure [12,13] for measuring the concentration dependence of the excess light scattering (R  ) at set angle  relative to the incident laser beam. However, results obtained by using this technique, termed composition gradient multi-angle light scattering (CG-MALS), on nonassociating proteins continue to be interpreted in terms of single-solute theory [14] and hence on the assumption that buffer components can be regarded as part of the solvent.…”