Determination of rate constants for rapid peroxidase reactions

Analytical Biochemistry
D C GoodwinT A Grover

Abstract

We describe an improved enzyme-monitored stopped-flow method for determining rate constants for peroxidase reactions that are too rapid to measure by conventional pseudo-first-order methods. Ascorbate will reduce many substrate radicals as rapidly as they are generated by a peroxidase. This ensures a constant substrate concentration during the peroxidase reaction. We investigated the reactions of horseradish peroxidase compound I (HRP-I) with three standard substrates (chlorpromazine (CPZ), 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), and p-methoxyphenol) in the presence and absence of ascorbate. The rate of reaction of CPZ with HRP-I is slow enough that it can be measured using pseudo-first-order conditions maintained by a minimum 10-fold excess of CPZ, or by the addition of ascorbate at very low CPZ concentrations. 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 concentrations 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(...Continue Reading

Citations

Oct 21, 2004·Biological Chemistry·María Angeles GilabertJosé Neptuno Rodríguez-López
Jun 7, 2014·Applied Biochemistry and Biotechnology·Zhiliang YuJianxun Lin
Jul 25, 2000·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·J KulysA Ziemys
Mar 17, 1998·Archives of Biochemistry and Biophysics·F E DayanN J Jacobs
Aug 19, 2003·Archives of Biochemistry and Biophysics·Douglas C Goodwin, Kristen M Hertwig
Jun 1, 2005·Langmuir : the ACS Journal of Surfaces and Colloids·Sekh MahiuddinWerner Kunz

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