The effect of 2,2-diphenyl-1-picrylhydrazyl and p-cresol on the oxidative degradation of indole-3-acetate

Abstract

A stable free radical compound, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and an oxidogenic substrate of peroxidase, p-cresol, both markedly stimulate the catalytic activity of 10 −8 m horseradish peroxidase in the oxidation of indole-3-acetate. However, at higher enzyme concentrations (10 −5 m), the oxidative degradation of the plant growth regulator is inhibited or prevented by these same compounds. The new finding that the overall effect of these agents is a function of enzyme concentration is interpreted in terms of the concentration-dependent reactivity of a reduced, oxygenated enzyme intermediate, compound III, in a second reaction with the promoting agents. Under some conditions the essential intermediate is destroyed rapidly in a second reaction with p-cresol or DPPH. However, formation of the enzyme compound is apparently accelerated at both high and low enzyme concentrations. IAA reacts directly with peroxidase and oxygen in an initial rapid reaction to cause accumulation of a mixture of reduced enzyme intermediates which exhibit absorbauce maxima near 418 and 430 mμ. However, oxygen is consumed in a second reaction which is presumed to be catalyzed by compound III (oxyferroperoxidase) which contains the bound superoxide anion. At low enzyme concentrations the first reaction is rate-limiting since it appears that compound III must accumulate before the oxygen-consuming reactions may proceed. Under these conditions, DPPH and p-cresol stimulate the initial process and the overall reaction. At higher enzyme levels the essential intermediate is formed more rapidly but is removed by the promoting agents leading to overall inhibition of IAA degradation.