β-Lactoglobulin B: A Proposed Standard for the Study of Reversible Self-Association Reactions in the Analytical Ultracentrifuge?

Abstract

Bovine β-lactoglobulin B is proposed for use as a standard in the measurement of reversible self-association reactions in the analytical ultracentrifuge. The protein is well understood on a molecular level, is readily obtainable, and is stable under harsh conditions. Bovine β-lactoglobulin B undergoes a simple monomer–dimer equilibrium which can be predictably controlled and consistently reproduced. In this investigation bovine β-lactoglobulin B has been studied via sedimentation equilibrium experiments in the XL-A analytical ultracentrifuge at 5–30°C in buffers of ionic strength 0.1–0.2Mand pH 2.0–3.0. Samples subjected to a number of different treatments and storage methods all yielded similar results. Molar equilibrium constants for the association reaction were determined by nonlinear regression fitting of a monomer–dimer model of association either to concentration versus radius data, using the programs NONLIN and ORIGIN, or to Ω versus concentration data using the program DUGOM. At 20°C and pH 2.6, over the ionic strength range 0.1–0.2M, the equilibrium constant for the association reaction ranges between 1 × 104and 5 × 104M−1. The parameters of nonideal self-association behavior were found to be independent of the particular analysis strategy. Fitting to the concentration distribution, the apparent weight-average molecular weight, or the Ω function all returned identical parameters.