Direct observation of the self-association of dilute proteins in the presence of inert macromolecules at high concentration via tracer sedimentation equilibrium: theory, experiment, and biological significance.
The technique of tracer sedimentation equilibrium [Rivas, G., et al. (1994) Biochemistry, 2341-2348 (1); Rivas, G., et al. (1996) J. Mol. Recognit. 9, 31-38 (2)] is utilized, together with an extension of the theory of sedimentation equilibrium of highly nonideal solutions [Chatelier and Minton, (1987) Biopolymers 26, 1097-1113 (3)], to characterize the thermodynamic activity and/or the state of association of a dilute, labeled macromolecular solute in the presence of an arbitary concentration of a second, unlabeled macromolecular solute. Experiments are performed on solutions of labeled fibrinogen (0.25-1 g/L) in bovine serum albumin (0-100 g/L) in the presence and absence of divalent cations (Ca(2+), Mg(2+)), and on solutions of labeled tubulin (0.2-0.6 g/L) in dextran (0-100 g/L). It is found that in the absence of the divalent cations, the large dependence of the thermodynamic activity of fibrinogen on BSA concentration is well accounted for by a simple model for steric repulsion. In the presence of the cations and sufficiently large concentrations of BSA (>30 g/L), fibrinogen appears to self-associate to a weight-average molar mass approximately twice that of monomeric fibrinogen. Tubulin appears to self-associate to an extent that increases monotonically with increasing dextran concentration, reaching a weight-average molar mass almost 3 times that of the alphabeta dimer in the presence of 100 g/L dextran. Possible biological ramifications are discussed.
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
G
G
Rivas
J A
JA
Fernandez
A P
AP
Minton
D002021:Buffers; D002413:Cations, Divalent; D003911:Dextrans / Q000737:chemistry; D019281:Dimerization; D004492:Edetic Acid; D005340:Fibrinogen / Q000737:chemistry; D006531:HEPES; D046911:Macromolecular Substances; D008954:Models, Biological; D008956:Models, Chemical; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012996:Solutions; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry; D014461:Ultracentrifugation
D002021:Buffers; D002413:Cations, Divalent; D003911:Dextrans; D046911:Macromolecular Substances; D012996:Solutions; D014404:Tubulin; D012710:Serum Albumin, Bovine; D005340:Fibrinogen; D004492:Edetic Acid; D006531:HEPES