A continuous fluorescence assay for tryptophan hydroxylase

A continuous fluorometric assay for tryptophan hydroxylase activity based o n the different spectral characteristics of tryptophan and 5-hydroxytryptop han is presented. Hydroxylation of tryptophan at the 5-position results in a large increase in the fluorescence of the molecule. The assay selectively monitors the fluorescence yield of 5-hydroxytryptophan by exciting the rea ction mix at 300 nm. The rate of increase of the emission signal was found to be directly proportional to the enzyme concentration. Inner filter effec ts due to quinonoid dihydropterin accumulation were eliminated by the inclu sion of a thiol reductant, Activity measured using this assay method was fo und to be the same as that determined by established discontinuous HPLC ass ay methods. The application of the assay to routine activity measurements a nd to steady-state determinations with the substrates tryptophan and tetrah ydropterin is described. (C) 1999 Academic Press.