Homovanillic acid as a fluorometric substrate for oxidative enzymes. Analytical applications of the peroxidase, glucose oxidase, and xanthine oxidase systems

Abstract

Fluorescence methods are described for the assay of the oxidative enzymes-peroxidase, glucose oxidase, and xanthine oxidase-based on the conversion of the nonfluorescent homovanillic acid to the highly fluorescent 2,2'-dlhydroxy-3,3'-dimethoxybiphenyl-5,5'-diacetic acid. The initial rate of formation of this fluorescent compound is measured and related to the ac-Recently, Guilbault, Kramer, and Hackley described the use of homovanillic acid (HVA) for the quantitative determination of peroxidase ( 14). The compound (I) is nonfluorescent, but is converted upon oxidation to the highly fluorescent compound (II), which has a Xex of 315 µ and a Xem at 425 µ. It was the intent of this study to develop fluorometric methods for a variety of oxidative enzyme systems using homovanillic acid, and using these new procedures, to develop specific and sensitive analytical methods

Changes in Ribonuclease Activity of Wheat Plants during Water Stress

“…Glucose oxidase and xanthine oxidase were assayed according to the method of Guilbault, et al (1968). The reaction mixture of glucose oxidase contained 2.7 ml of 0.1 M Tris-HCl buffer, pH 8.1, 0.1 ml of 0.1 M glucose, 0.1 ml of 2.5 mg/ml homovanillic acid, 0.1 ml of 0.75 mg/ml peroxidase and 0.1 ml of glucose oxidase (15 ~g/ml).…”

Sistema antioxidante envolvendo o ciclo metabólico da glutationa associado a métodos eletroanalíticos na avaliação do estresse oxidativo

“…Para exemplificar, algumas enzimas que foram usadas para tais aná-lises incluem glutationa peroxidase 87,88 , "horseradish" peroxidase 89 e ciclooxigenase 90 . Citocromo c 91 e microperoxidase 92 têm sido aplicadas na detecção de hidroperóxidos por quimiluminescência combinada com HPLC.…”

A Novel Method for Determining Peroxidase Activities Using p-Acetamidophenol Analogs

“…To 10 ml THE solution of 5,5'-diamino-2,2'-dimethoxybiphenyl (100 mg, 0.409 mmol), acetic anhydride (116 µl, 1.13 mmol) and triethylamine (112 p1, 0.82 mmol) were added; the solution was stirred at ambient temperature for 1 h. The resulting white precipitate was collected and washed with THE to afford 125 mg (93%) of 5,5'-diacetamido-2,2'-dimethoxybiphenyl as a white powder. In a fluorescent assay study using p-acetamidophenol analogs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), it was found that contamination from the parent p-aminophenols caused undesirable effects, such as inducing a time lag in the enzymatic reacton with HRP at low HRP activity and an instability from the stock solutions ofp-acetamidophenols.…”