Quantitative evaluation of solution equilibrium binding interactions by affinity partitioning: application to specific and nonspecific protein-heparin interactions.
A variation of the quantitative affinity chromatography (QAC) method of Winzor, Chaiken, and co-workers for the analysis of protein-ligand interactions has been developed and used to characterize sequence-specific and nonspecific protein-heparin interactions relevant to blood coagulation. The method allows quantitation of the binding of two components, A and B, from the competitive effect of one component, B, on the partitioning of the other component, A, between an immobilized acceptor phase and solution phase at equilibrium. Under the conditions employed, the differences in total A concentrations yielding an equivalent degree of saturation of the immobilized acceptor in the absence and presence of B defines the concentration of A bound to B in solution, thereby enabling conventional Scatchard or nonlinear least-squares analysis of the A-B equilibrium interaction. Like the QAC method, quantitation of the competitor interaction does not depend on the nature of the affinity matrix interaction, which need only be described empirically. The additional advantage of the difference method is that only the total rather than the free competitor ligand concentration need be known. The method requires that the partitioning component A be univalent, but allows for multivalency in the competitor, B, and can in principle be used to study binding interactions involving nonidentical, interacting, or nonspecific overlapping sites. Both the binding constant and the stoichiometry for the specific antithrombin-heparin interaction as well as the apparent binding constant for the nonspecific thrombin-heparin interaction at low thrombin binding densities obtained using this technique were in excellent agreement with values determined using spectroscopic probes.
Olson
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Henry Ford Hospital, Division of Biochemical Research, Detroit, Michigan 48202.
Bock
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Sheffer
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D000990:Antithrombin III / Q000378:metabolism; D001667:Binding, Competitive; D002846:Chromatography, Affinity / Q000379:methods*; D006493:Heparin / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012996:Solutions; D013917:Thrombin / Q000378:metabolism*
D011506:Proteins; D012996:Solutions; D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin