Inhibition of the Peroxidase-Catalyzed Oxidation of Chromogenic Substrates by Alkyl-Substituted Diatomic Phenols

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A comparative kinetic study of the peroxidase oxidation of three chromogenic substrates—2,2′-azino-bis(3-ethyl-2,3-dihydrobenzothiazoline-6-sulfonic acid), o-phenylenediamine (PDA), and 3,3′,5,5′-tetramethylbenzidine—inhibited by trimethylhydroquinone and six tert-butylated pyrocatechols (InH) was carried out at 20°C in 0.015 M phosphate–citrate buffer (pH 6.0) containing organic cosolvents (0–10% ethanol or DMF). The inhibitors were quantitatively characterized by the inhibition constants (Ki), the duration of the lag period in the oxidation product formation (Δτ), and the stoichiometric coefficient of inhibition that specifies the number of radicals terminated by one InH molecule (f). The inhibition could be competitive, noncompetitive, mixed, or uncompetitive, which depended on the nature and structure of the chromogenic substrate–diatomic phenol pair. Various substrate–diatomic phenol pairs exhibited Ki values within the range of 11–240 μM and f values from 0.7 to 2.6. The absence of a lag period was characteristic of oxidation of the substituted o-phenylenediamine–substituted pyrocatechol. The total kinetic parameters and properties of the components allowed us to suggest six chromogenic substrate–substituted diatomic phenol pairs for use in test systems for the determination of antioxidant activity in human body fluids, natural biological preparations, and food.

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