Beta-lactoglobulin B: a proposed standard for the study of reversible self-association reactions in the analytical ultracentrifuge?
Bovine beta-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 beta-lactoglobulin B undergoes a simple monomer-dimer equilibrium which can be predictably controlled and consistently reproduced. In this investigation bovine beta-lactoglobulin B has been studied via sedimentation equilibrium experiments in the XL-A analytical ultracentrifuge at 5-30 degrees C in buffers of ionic strength 0.1-0.2 M and 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 Omega versus concentration data using the program DUGOM. At 20 degrees C and pH 2.6, over the ionic strength range 0. 1-0.2 M, the equilibrium constant for the association reaction ranges between 1 x 10(4) and 5 x 10(4) M-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 Omega function all returned identical parameters.
Joss
L A
LA
Department of Biochemistry, University of Sydney, Sydney, New South Wales, 2006, Australia.
Ralston
G B
GB
D000818:Animals; D002417:Cattle; D007782:Lactoglobulins / Q000737:chemistry*; D008970:Molecular Weight; D009994:Osmolar Concentration; D011485:Protein Binding; D013816:Thermodynamics; D014461:Ultracentrifugation / Q000592:standards*
D007782:Lactoglobulins