TRANSIENT AND STEADY-STATE KINETICS OF THE OXIDATION OF SCOPOLETIN BYHORSERADISH-PEROXIDASE COMPOUND-I, COMPOUND-II AND COMPOUND-III IN THE PRESENCE OF NADH

Scopoletin, a naturally occurring fluorescent component of some plants and a proven plant growth inhibitor, is a known reactant with peroxid ase. However, the kinetics of the elementary steps of the reaction hav e never been investigated, nor has the quantitative effect of interfer ing substances ever been explored in detail, despite the fact that sco poletin is widely used in a peroxidase assay for H2O2. In this work, w e 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 II: (3.7 +/ - 0.1) X 10(6) M(-1) s(-1) and (8.5 +/- 0.5) X 10(5) M(-1) s(-1) at 20 degrees C, pH 6.0 and ionic strength of 0.1 M. We investigated the po ssible inhibitory effect of NADH on the reaction of scopoletin with HR P and also the effect of scopoletin on the NADH reaction. In the prese nce of NADH the rate constant for the reaction between HRP-I and scopo letin decreased slightly to (2.8 +/- 0.1) X 10(6) M(-1) s(-1). Thus, a lthough NADH is also a peroxidase substrate, it cannot compete effecti vely for the oxidized forms of the enzyme. On the other hand, scopolet in stimulates the oxidation of NADH by the HRP/H2O2 system, apparently by forming a phenoxyl radical which then oxidizes NADH to NAD' radica ls. We present spectral evidence showing that in the aerobic reaction between HRP and NADH at pH 7.0 (without exogenously added H2O2) HRP-II is the dominant enzyme intermediate with HRP-III also detectable. Add ition of scopoletin to the HRP/ NADH system leads to a biphasic reacti on in which HRP-II and HRP-III disappear. The rate constants for both phases are linearly dependent on scopoletin concentration. We attribut e the faster phase to the HRP-II reaction with scopoletin with a rate constant of (6.2 +/- 0.1) X 10-(5) M(-1) s(-1) and the slower phase to the HRP-III reaction with scopoletin with rate constant (5.0 +/- 0.4) X 10(4) M(-1) s(-1). Our present work not only provides rate constant s for the oxidation of scopoletin by HRP-I, II and III but also elucid ates the interactions that possibly occur physiologically during NADH oxidation in the presence of scopoletin.