Solid-phase assay for determination of binding parameters of ligand-protein complexes with high dissociation rates.
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.
Santa-Coloma
T A
TA
Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina.
Rossi
R J
RJ
Charreau
E H
EH
D000818:Animals; D002022:Bufo arenarum; D002352:Carrier Proteins / Q000378:metabolism*; D002606:Charcoal; D002848:Chromatography, DEAE-Cellulose / Q000379:methods*; D005260:Female; D012739:Gonadal Steroid Hormones / Q000378:metabolism*; D006207:Half-Life; D007700:Kinetics; D008024:Ligands; D012738:Sex Hormone-Binding Globulin
D002352:Carrier Proteins; D012739:Gonadal Steroid Hormones; D008024:Ligands; D012738:Sex Hormone-Binding Globulin; D002606:Charcoal