Collect. Czech. Chem. Commun. 1990, 55, 2152-2160

Phenol-sensitive enzyme electrode with substrate cycling for quantification of certain inhibitory aromatic acids and thio compounds

Lumír Macholán

Department of Biochemistry, Masaryk University, 611 37 Brno


A Clark oxygen electrode coated with a membrane of crosslinked champignon phenol oxidase (tyrosinase)is able to detect phenol containing analytical samples 3-5 times more sensitively after the admixture of hydrazine hydrochloride to the reaction buffer. This compound will recycle the substrate via chemical reduction of the quinoid product thus simultaneously preventing the membrane from rapid blackening. The decrease of oxygen in the membrane due to phenol oxidation is rapidly compensated by diffusion from the bulk solution when an inhibitor of the membrane-bound phenol oxidase is added to the reaction medium. This effect has been utilized for the measurement of low concentrations of compounds decreasing the sensitivity of the bioelectrode either reversibly (benzoic acid, 3- and 4-aminobenzoic acid, salicylic acid, 4-aminosalicylic acid, nicotinic acid, 4-nitrophenol, 1-naphthol) or irreversibly (phenylthiourea, thioacetamide, L-cysteine, reduced glutathione, 2-mercaptoethanol, 2,3-dimercaptopropanol and others). Benzoate as a potent reversible inhibitor markedly stabilizes the phenol oxidase reaction membrane during its storage either in the buffer or in dry state at 4 °C for one year at least.