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 kinetic analysis of sensorgrams which deviate from the pseudo-first-order kinetic behavior predicted for 1:1 interactions between ligate and affinity sites on the sensor surface is described. This analysis employs quantitative expressions that are used in conventional kinetic characterization of protein interactions by biosensor technology, but with the equilibrium sensorgram response fixed at a predetermined magnitude. Simulated sensorgrams for situations in which the aberrant kinetic behavior reflects (i) heterogeneity of affinity sites and (ii) isomerization of the complex between ligate and affinity sites are used to explore the feasibility of the approach. Its application is then illustrated with BIAcore studies of the interaction between the Fab fragment of an anti-paraquat monoclonal antibody and immobilized antigen in the form of a paraquat analog attached covalently to the sensor surface. Studies with an extremely high degree of antigen substitution on the sensor surface yielded sensorgrams that deviated markedly from pseudo-first-order kinetic behavior. However, they yielded the same binding constant (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 surface. Such identity of binding constants eliminates heterogeneity of immobilized paraquat sites as the likely source of the aberrant kinetic behavior.
Bowles
M R
MR
Department of Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, Australia.
Hall
D R
DR
Pond
S M
SM
Winzor
D J
DJ
D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D015374:Biosensing Techniques; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007700:Kinetics; D010269:Paraquat / Q000276:immunology; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D011506:Proteins; D010269:Paraquat