Abstract
Publisher Summary This chapter discusses the catalytic assay method used for the analysis of diphosphopyridine nucleotide (DPN). A new catalytic assay for DPN is developed that improves on the weak points of the older assay. The pyridine nucleotide to be measured is shared as coenzyme by a pair of dehydrogenases in the assay method. In a cyclic process, the DPN alternately oxidizes the substrate of one enzyme and reduces that of the other. For each mole of DPN, more than 10,000 moles of each product is produced in an hour. Since the nucleotide concentration is kept far below the Michaelis constants, the rate of product accumulation is proportional to pyridine nucleotide concentration. Accordingly, the amount of either product formed can be used as a measure of the pyridine nucleotide. The reagents used for cycling step, indicator step, standard solutions, and source of reagents are also discussed. There are special applications of the cycling assay for DPN, in which the use of glutamate as an indicator may lead to erroneous results. The method is also useful for the enzymatic analysis of prostaglandins.
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