STUDIES OF PROTEIN INTERACTIONS BY BIOSENSOR TECHNOLOGY - AN ALTERNATIVE APPROACH TO THE ANALYSIS OF SENSORGRAMS DEVIATING FROM PSEUDO-FIRST-ORDER KINETIC-BEHAVIOR

A procedure for evaluating the thermodynamic equilibrium constant by k inetic analysis of sensorgrams which deviate from the pseudo-first-ord er kinetic behavior predicted for 1:1 interactions between ligate and affinity sites on the sensor surface is described. This analysis emplo ys quantitative expressions that are used in conventional kinetic char acterization of protein interactions by biosensor technology, but with the equilibrium sensorgram response fixed at a predetermined magnitud e. Simulated sensorgrams for situations in which the aberrant kinetic behavior reflects (i) heterogeneity of affinity sites and (ii) isomeri zation of the complex between ligate and affinity sites are used to ex plore the feasibility of the approach. Its application is then illustr ated with BIAcore studies of the interaction between the Fab fragment of an antiparaquat monoclonal antibody and immobilized antigen in the form of a paraquat analog attached covalently to the sensor surface. S tudies with an extremely high degree of antigen substitution on the se nsor surface yielded sensorgrams that deviated markedly from pseudo-fi rst-order kinetic behavior. However, they yielded the same binding con stant (3 x 10(6) M(-1)) as the value deduced by conventional analysis of sensorgrams that conformed with pseudo-first-order kinetics because of a much lower concentration of immobilized antigen on the sensor su rface. Such identity of binding constants eliminates heterogeneity of immobilized paraquat sites as the likely source of the aberrant kineti c behavior. (C) 1997 Academic Press, Inc.