Proposed Model for the Peroxidase-Catalyzed Oxidation of Indole-3-acetic Acid in the Presence of the Inhibitor Ferulic Acid

Abstract

Linear increments in ferulic acid concentration produce logarithmic increases in the ferulic acid-induced lag periods prior to the peroxidase-catalyzed oxidation of indole-3-acetic acid in a system containing 2,4-dichlorophenol and MnCL in acetate buffer at pH 5.6. Maintaining the ratio of indole-3-acetic acid to ferulic acid constant at 100 while linearly raising the ferulic acid concentration results in linear increases in the lag period. Both indole-3-acetic acid and ferulic acid are substrates of horseradish peroxidase in the presence of H202, and indole-3-acetic acid competitively inhibits the oxidation of ferulic acid. A model for the enzymatic oxidation of indole-3-acetic acid catalyzed by peroxidase is proposed.It is well established that plants contain enzymes (5,11,15,(18)(19)(20)21). Most inhibitors are of low molecular weight, although Stonier et al. (18,20) reported phenolic inhibitors from Pharbitis with molecular weights of 5,000 to 10,000, based upon behavior on sephadex gels. The association of high inhibitor concentrations with actively growing tissues suggests that inhibitors promote growth by preventing IAA destruction by endogenous IAA oxidase-the "auxin protector" concept of Stonier and Yoneda (20).Several workers have sought to clarify the mechanism(s) by which phenolic inhibitors delay the peroxidase-catalyzed oxidation of IAA (15,(18)(19)(20)21 Gordon and Weber (6). Two ml of test solution were mixed with 4 ml of reagent, and absorbance was measured at 525 nm on a Coleman 46 spectrophotometer equipped with a Gilson transferator. Color development was allowed to occur in darkness for about 1 hr before measuring absorbance.DMACA reagent prepared according to the method of Meudt and Gaines (9) was used to measure the accumulation of IAA oxidation products. Two ml of test solution were added to 2 ml of reagent, and the samples were allowed to stand in darkness about 1 hr before absorbance at 562 nm was determined as described above.Comparison Experiment. An experiment was designed to permit simultaneous measurements of changes in IAA, FA, and IAA oxidation products during the course of the reaction. A beaker containing the following concentrations of reactants in a final volume of 180 ml was allowed to equilibrate for about 1 hr in a water bath at 30 C: 0.1 mm IAA, 0.1 mm DCP, 0.1 mM MnCl,, 7.0 uM FA, and 50 mm acetate buffer, pH 5.6. To start the reaction, the beaker was placed on a magnetic stirrer and 30 ml of ice cold HRP was poured in, producing a final HRP concentration of 0.5 ,ug/ml. An accurate elapsedtime clock and the spectrophotometer recorder were started the moment the HRP was added and allowed to run throughout the experiment (25 min). Immediately after adding the HRP, the beaker was returned to the water bath for the duration of 967 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

Physiological Aspects of Free-Radical Reactions

“…The mechanism of oxidation of a plant hormone, IAA has been a subject of investigation by many plant physiologists (126)(127)(128)(129)(130)(131)(132)(133)(134)(135). It is now accepted that free radical species which are formed through catalysis of peroxidase are intermediates in the degradation of IAA (128)(129)(130)(131)(132)(133)(134).…”

The Reaction of Coumarins with Horseradish Peroxidase

“…The peroxidase-catalyzed oxidation of hydroxylated derivatives of cinnamic acid has been widely studied (6,8,13,18) due to the implication of these compounds in the metabolism of IAA (1, 10). Horseradish peroxidase peroxidizes coumarins such as scopoletin and other cinnamic acid-derived phenols such as ferulic acid (8).…”

“…In this second pathway a reductant, IAA, and molecular oxygen initiate the oxidation of scopoletin. Moreover, it has been shown (8,18) that the rapid reaction between phenolic inhibitors and an activated enzyme intermediate induces a lag in the course of IAA cooxidation. A previous study (18) of the rate of oxidation of IAA during the lag period showed that scopoletin competitively inhibits the catalytic oxidation of IAA even though IAA is required for initiation of the oxidation of the inhibitors.…”

“…Horseradish peroxidase peroxidizes coumarins such as scopoletin and other cinnamic acid-derived phenols such as ferulic acid (8). The stoichiometric oxidation of scopoletin by free H202 in the presence of HRP2 often has been used as an analytical method for determining H202 (2,13).…”

“…The differences observed between the two scopoletin oxidation pathways reflect: (a) the competition between indole-3-acetate and scopoletin for peroxidase compounds; (b) the lower reactivity of scopoletin with peroxidase compound III compared with peroxidase compound II. The peroxide-promoted reaction is eliminated by catalase, while the indole-3-acetate initiated oxidation is not affected by excess quantities of either catalase or superoxidase dismutase.The peroxidase-catalyzed oxidation of hydroxylated derivatives of cinnamic acid has been widely studied (6,8,13,18) due to the implication of these compounds in the metabolism of IAA (1, 10). Horseradish peroxidase peroxidizes coumarins such as scopoletin and other cinnamic acid-derived phenols such as ferulic acid (8).…”