Solid phase assay for determination of binding parameters of ligand protein complexes with high dissociation rates

SANTA-COLOMA TA; CHARREAU EH; ROSSI RJ

ANALYTICAL BIOCHEMISTRY

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 1991

0003-2697

The binding parameters, the affinity constant (Ka) and binding capacity (Q), of a protein possessing ligand-protein complexes with a high dissociation rate (Sex Steroid Binding protein from Bufo arenarum) were determined using a solid-phase method. The technique is based upon the adsorption of the steroid-protein complex to DEAE-cellulose. This method was compared with a nonequilibrium method (charcoal adsorption of free ligand), and the latter resulted in underestimation of both binding parameters, Ka and Q. The solid-phase method reported here is appropriate to determine the binding parameters of proteins with high dissociation rates because the results are independent of the complex half-time. The method also possesses advantages compared to other equilibrium assays such as dialysis or steady-state electrophoresis. With minor modifications, it may be useful to characterize different proteins, particularly those possessing ligand-protein complexes with very high dissociation rates.