THEORETICAL AND EXPERIMENTAL CONSIDERATIONS OF THE PSEUDO-FIRST-ORDERAPPROXIMATION IN CONVENTIONAL KINETIC-ANALYSIS OF IASYS BIOSENSOR DATA

The validity of the conventional interpretation of IAsys biosensor pro files in terms of pseudo-first-order kinetic behavior is subjected to closer scrutiny by its application to simulated data for low- and high -affinity interactions between ligate and immobilized ligand. As might reasonably have been expected, analysis of the simulated data for the low-affinity system (association equilibrium constant of 10(5) M-1) i n such terms returned the input association and dissociation rate (10( 3) M-1 s(-1) and 10(-2) s(-1), respectively)-a consequence of essentia l compliance with the assumed constancy of ligate concentration in the liquid phase. For the high-affinity interaction (k(a) = 10(5) M-1 s(- 1), k(d) = 10(-2) s(-1), K-AX = 10(7) M-1) the ligate concentration wa s depleted by up to 35%, and hence its assumed constancy was clearly a n untenable approximation. Whereas no symptomatic evidence of such vio lation (apart from the return of incorrect estimates of k(a) and k(d)) was evident from pseudo-first-order kinetic analysis of the adsorptio n profiles, the corresponding analysis of desorption profiles was more informative in that the data deviated demonstrably from pseudo-first- order kinetic behavior. A second-order kinetic analysis was therefore developed and shown to be applicable to adsorption and desorption prof iles, irrespective of the validity or otherwise of the pseudo-first-or der kinetic approximation. Experimental results obtained for the inter action of histidine-rich glycoprotein with immobilized IgG were then u sed to illustrate various features of the pseudo-first order and secon d-order kinetic analyses, and to determine from the second-order analy sis an association equilibrium constant of 2 x 10(8) M-1, which is 20- fold greater than the value obtained by interpretation of the profiles in terms of pseudo-first-order kinetic behavior. (C) 1997 Academic Pr ess.