DETERMINATION OF RATE CONSTANTS FOR RAPID PEROXIDASE REACTIONS

We describe an improved enzyme-monitored stopped-flow method for deter mining rate constants' for peroxidase reactions that are too rapid to measure by conventional pseudo-first-order methods. Ascorbate will red uce many substrate radicals as rapidly as they are generated by a pero xidase. This ensures a constant substrate concentration during the per oxidase reaction. We investigated the reactions of horseradish peroxid ase compound I (HRP-I) with three standard substrates (chlorpromazine (CPZ), 2,2'-azino - bis[3 - ethylbenzthiazoline - 6- sulfonic acid] (A BTS), and p-methoxyphenol) in the presence and absence of ascorbate. T he rate of reaction of CPZ with HRP-I is slow enough that it can be me asured using pseudo-first-order conditions maintained by a minimum 10- fold excess of CPZ, or by the addition of ascorbate at very low CPZ co ncentrations. The same rate constant was obtained with either method. The rate of reaction of ABTS with HRP-I at lower pH (5.0-3.3) is rapid ; consequently, we were unable to obtain rate constants using concentr ations of ABTS which constitute pseudo-first-order conditions. However , using much lower ABTS concentrations with the addition of ascorbate, we obtained rate constants that ranged from 5 x 10(7) to 8 x 10(8) M( -1) s(-1). Hence, ascorbate provides a simplified way to maintain pseu do-first order conditions for fast peroxidase reactions even at low su bstrate concentrations. (C) 1995 Academic Press, Inc.