Effect of Strophanthin G on Peroxidase Oxidation Kinetics of Slowly Oxidizable Peroxidase Substrates

V. V. Rogozhin^* and T. V. Rogozhina

Yakutsk State Agricultural Academy, ul. Krasil'nikova 15, Yakutsk, 677002 Russia

^* To whom correspondence should be addressed.

Received January 10, 1999; Revision received June 20, 1999

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Steady-state kinetics of thioproperazine, triftazine, aminazine, and o-dianisidine oxidation with hydrogen peroxide catalyzed by horseradish peroxidase were studied in the presence of strophanthin G. Values of the inhibition and activation constants (K_i, K_a) were determined in the pH range 5.0-7.5. At acidic pH, strophanthin G activated peroxidase during the oxidation of thioproperazine by the uncompetitive mechanism, and when triftazine was oxidized, the inhibition was noncompetitive. At pH > 6.0, the patterns of activation and inhibition changed to mixed-type during the peroxidase oxidation of thioproperazine and triftazine and to competitive inhibition of peroxidase with strophanthin G during the oxidation of aminazine. These effects are suggested to be due to an ionizable enzyme group of pK ~ 6.0. Strophanthin G inhibited free-radical oxidation of o-dianisidine via binding to the enzyme-substrate complex, preventing the generation of a stable semi-oxidized product of o-dianisidine, and thus inhibiting the enzyme by the anticompetitive mechanism. Mechanisms of oxidation of slowly and rapidly oxidizable substrates of peroxidase in the presence of strophanthin G are suggested.

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KEY WORDS: horseradish peroxidase, 2-chloro-10-(3-dimethylaminopropyl)phenothiazine, 3-fluoromethyl-10-{3-(1-methylpiperazinyl-4)propyl}phenothiazine, 2-dimethylsulfamido-10-{3-(1-methylpiperazinyl-4)propyl}phenothiazine, strophanthin G, o-dianisidine, antioxidants

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