EFFECTS OF MIXED-SOLVENTS ON 3 ELEMENTARY STEPS IN THE REACTIONS OF HORSERADISH-PEROXIDASE AND LACTOPEROXIDASE

The effects of methanol, acetone, and ethylene glycol (up to 50%, v/v) on elementary steps in the reactions of horseradish peroxidase (HRP) and lactoperoxidase (LPO) were studied by means of the stopped-flow me thod and the difference spectrum. The rate constant (k(3,app)) of the oxidation reaction of p-cresol with HRP compound II was remarkably red uced in the presence of organic solvents (to 2.3%, 1.8% and 9.4% of th e original value in the presence of 50% (v/v) of methanol, acetone and ethylene glycol, respectively), then to a lesser degree were decrease d the rate of the oxidation reaction with LPO compound II, and the rat e of the oxidation reaction with HRP compound I. These reductions in t he reaction rates were not due to competitive inhibition of the solven ts, but considered to be related to the degree of exposure of the elec tron transfer route to the medium. While the rate constant of compound I formation (k(1,app)) was moderately affected by organic solvents in the case of HRP, the reaction rate with LPO was scarcely affected by organic solvents, being in harmony with the compact heme crevice which probably hampers penetration of solvent molecules. The rate constant (k(2,i,app)) of the oxidation reaction of an iodide ion by HRP compoun d I was also hardly affected by the solvents. On the basis of these fa cts, the mechanism of electron transfer from donors to compound I and compound II is discussed.