Transient and Steady‐State Kinetics of the Oxidation of Scopoletin by Horseradish Peroxidase Compounds I, II and III in the Presence of NADH

Abstract

Scopoletin, a naturally occurring fluorescent component of some plants and a proven plant growth inhibitor, is a known reactant with peroxidase. However, the kinetics of the elementary steps of the reaction have never been investigated, nor has the quantitative effect of interfering substances ever been explored in detail, despite the fact that scopoletin is widely used in a peroxidase assay for H,O,. In this work, we employed both transient-state and steady-state methods to determine the second-order rate constants for the oxidation of scopoletin by the horseradish peroxidase (HRP) intermediate compounds I and 11: (3.7-t0.1)X10h M~' s I and (8.5-+0.5)X1Os M --' s .I at 20°C, pH 6.0 and ionic strength of 0.1 M. We investigated the possible inhibitory effect of NADH on the reaction of scopoletin with HRP and also the effect of scopoletin on the NADH reaction. In the presence of NADH the rate constant for the reaction between HRP-I and scopoletin decreased slightly to (2.820.1)XlO" M I s -' . Thus, although NADH is also a peroxidase substrate, it cannot compete effectively for the oxidized forms of the enzyme. On the other hand, scopoletin stimulates the oxidation of NADH by the HRP/H,O, system, apparently by forming a phenoxyl radical which then oxidizes NADH to NAD' radicals. We present spectral evidence showing that in the aerobic reaction between HRP and NADH at pH 7.0 (without exogenously added HZOJ HRP-I1 is the dominant enzyme intermediate with HRP-I11 also detectable. Addition of scopoletin to the HRP/ NADH system leads to a biphasic reaction in which HRP-I1 and HRP-I11 disappear. The rate constants for both phases are linearly dependent on scopoletin concentration. We attribute the faster phase to the HRP-I1 reaction with scopoletin with a rate constant of (6.2f0.1)X10s M -' s~-' and the slower phase to the HRP-I11 reaction with scopoletin with rate constant (5.0t0.4)X104 M ' s '. Our present work not only provides rate constants for the oxidation of scopoletin by HRP-I, I1 and 111 but also elucidates the interactions that possibly occur physiologically during NADH oxidation in the presence of scopoletin.Keywords: scopoletin; NADH ; horseradish peroxidase; transient-state kinetics; steady-state kinetics. Scopoletin (7-hydroxy-6-methoxy-2H-l-benzopyran-2-one)is a pharmacologically active agent that has been isolated from several plant species [I -31. It contributes to the blue fluorescence of cotton leaves [4, 51 and proves to be the most active of all coumarin derivatives in causing inhibition of root growth 161. Its localization in plant cells and its ability to serve as an aromatic donor molecule suggest that it could be a physiological substrate of the ubiquitous peroxidase enzymes. Scopoletin has been found to be the most potent naturally occurring stimulator of indoleacetic acid oxidase activity 171. Since phenolic compounds are known enhancers of peroxidase-catalyzed oxidations [8], we were interested not only in scopoletin's behavior as a peroxidase substrate but also how it affects the oxidation o...

Oxidation of guaiacol by myeloperoxidase: a two-electron-oxidized guaiacol transient species as a mediator of NADPH oxidation

“…The addition of glucose-6-phosphate dehydrogenase to the spent reaction mixture regenerated nearly all of the added NADPH (Figure 9, curve d), confirming that NADP + was the only oxidized compound produced from NADPH. Similar effects were reported for the cooxidation of NAD(P)H by the HRP\H # O # system mediated by phenoxyl radicals from thyroxine, various arylamine or phenolic substrates, paracetamol and scopoletin [19,20,34,35].…”

“…As expected from previous reports on NAD(P)H co-oxidation [19,20,[33][34][35], O # − d might be involved in the propagation of NADH oxidation by peroxidases. To further investigate the mechanism of reaction, and the participation of O # − d in the oxidation of NADPH, we examined the effects of the addition of SOD on the time courses of both guaiacol and NADPH oxidation (Figure 8, upper panel, curves a and b).…”

“…It is well established that the oxidation of NAD(P)H by HRP\H # O # under steady-state conditions requires the presence of a mediating molecule that acts as a redox catalyst for NAD(P)H oxidation [18][19][20][33][34][35] Abbreviations used : Tyr-OH, tyrosine ; Tyr-Od, tyrosyl radical ; NADPd, NADP radical ; C I and C II, compounds I and II respectively. NADPH oxidation is totally dependent on the turnover of MPO.…”

Mitochondrial α-Ketoglutarate Dehydrogenase Complex Generates Reactive Oxygen Species

“…However, it was found that although NADH can react with the oxidized form of HRP, it cannot effectively compete with scopoletin in an HRP-scopoletin H 2 O 2 assay (Marquez and Dunford, 1995). Furthermore, rat brain mitochondria contain ϳ5 nmol of total NAD/mg protein (C. Chinopoulos, unpublished observations) similar to the amount found in mitochondria from other tissues (3-7 nmol of total NAD/mg mitochondrial protein) (Tischler et al, 1977;Di Lisa et al, 2001).…”

Chorion peroxidase-mediated NADH/O2 oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H2O2 formation during chorion hardening in Aedes aegypti mosquitoes

“…These enzymes occupy a special position in biochemistry and enzymology and are among the most extensively studied enzymes. In addition to their primary peroxidative reaction cycle with H 2 O 2 as oxidizing agent, some peroxidases show an oxidase activity mediating the reduction of O 2 to superoxide ( ) and H 2 O 2 by substrates such as epinephrine, dihydroxyfumarate, reduced nicotinamide adenine dinucleotide (NADH) and indole-3-acetic acid [1][2][3][4][5][6][7]. There are also reports that discuss the catalase activity of the peroxidases [8,9], especially from fungi [10][11][12].…”