Abstract
Abstract An enzymatic microdetermination method is described to determine the concentration of biologically active hydroxylase cofactor in crude samples. A small amount of hydroxylase cofactor was oxidized and reduced in a cyclic fashion by phenylalanine 4-monooxygenase (EC 1.14.16.1, PheOHase) and dihydropteridine reductase (EC 1.6.99.7, DPRase) in the presence of excess phenylalanine (Phe) and NADH. The amount of NAD+, accumulated as a result of this cyclic reaction (first cycling), was in proportion to that of the hydroxylase cofactor. The NAD+ was further amplified by an enzymatic amplification reaction, NAD cycling (second cycling) and determined fluorometrically. This double cycling method with more than a 20,000 fold amplification provided extremely high sensitivity, down to 0.02 pmol per assay, and was successfully applied to crude microsamples of pg wet weight.
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