The Reaction of Coumarins with Horseradish Peroxidase

Abstract

The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horseradish peroxidase has been investigated.Peroxidase catalyzed coumarin oxidation requires either an electron donor and molecular oxygen or hydrogen peroxide. If peroxide is present, the reaction is mediated by peroxidase compound II which reacts rapidly and stoichiometrically with scopoletin. Different oxidation products are formed, depending on whether IAA or hydrogen peroxide promotes the reaction. A scopoletin-free radical intermediate has been isolated from the peroxide reaction mixture but was not detected in the peroxide-free system.When indole-3-acetate is the electron donor, reduced peroxidase combines with molecular oxygen to give peroxidase compound III. Added scopoletin is cooxidized with indole-3-acetate. Compared to the scopoletin peroxidation, this reaction is slower and yields fewer coumarin oxidation products. 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). 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). This reaction must proceed through the mediation of peroxidase compounds I and II. The complex mixture of products of the reaction has not been previously investigated. Evidence is presented here for the oxidation state of the initial product and for possible structures of other products.Coumarin oxidation can also proceed in the absence of H202. 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. This inhibition was found to be of a non-linear type since the plots of rec...

The Versatility of Antioxidant Assays in Food Science and Safety—Chemistry, Applications, Strengths, and Limitations

“…The formation of this intermediate results from the loss of the blue fluorescence, since scopoletin is being consumed in the reaction. The blue intermediate is slowly oxidized into an insoluble yellow complex (C 7 H 8 O 3 ) on standing, which can be read either at 417–402 nm or at 385 nm, depending on different studies [ 63 , 75 , 76 ]. However, the addition of a potential AO will prevent the oxidation process to occur, thus, hindering the formation of the yellow complex.…”

“…When [63,75,76]. However, the addition of a potential AO will prevent the oxidation process to occur, thus, hindering the formation of the yellow complex.…”

Cascade DNA logic device programmed ratiometric DNA analysis and logic devices based on a fluorescent dual-signal probe of a G-quadruplex DNAzyme

“…This fluorescent substrate exhibits fluorescence emission at 465 nm and could be oxidized to a non-fluorescent product with unknown structure by H 2 O 2 catalysed by HRP or G4zyme. 14 The mechanism of G4zyme to Sc/H 2 O 2 is presented in Scheme 1A. 12 To testify the feasibility of our strategy, verification experiments were explored, strand 1G3 was integrated with GGGTAGGGCGGG at the 3 0 terminus and strand 1G1 was integrated with TGGGT at the 5 0 terminus were used as two hybridized strands which could form a G-quadruplex.…”

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“…Scopoletin has been found in several plants (Reppel and Wageobreth 1958, Letham 1978, Brown 1981, Murray et al 1982. In vitro it protects indole-3-acetate against oxidation by peroxidases (Miller et al 1975) and it might therefore have a regulatory role in plant growth.…”