Use of a fluorogenic or chromogenic peroxidase enzyme substrate to detect phosphate

摘要

A method for the determination of inorganic phosphate which comprises the use of coupled enzyme reactions to generate hydrogen peroxide which analyte is then detected with fluorogenic or chromogenic peroxidase enzyme substrates. The method involves selecting a phosphorylase enzyme and oxidase enzyme in combination so that, in the presence of inorganic phosphate, the two enzymes produce hydrogen peroxide. Example combinations are: maltose phosphorylase/glucose oxidase, trehalose phosphorylase/glucose oxidase, purine nucleoside phosphorylase/xanthine oxidase. Generation of hydrogen peroxide is detected using derivatives of dihydroxanthenes or phenoxazines e.g. dihydrofluoresceins (=fluorescins), dihydrorhodamines and 10-acetyl-3,7-dihydroxyphenoxazine (= Amplex Red), dihydroresorufin that are colourless or nonfluorescent until oxidised by the peroxidase enzyme. Other analytes may be detected using this method e.g. ATPase, alkaline phosphatase, maltose, protein phosphatases, apyrase, adenylyl cyclase, prenyl transferase, pyrophosphatase, and 5'-nucleotidase.