A continuous fluorescence assay for tryptophan hydroxylase

Graham R. Moran, Paul F. Fitzpatrick

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

A continuous fluorometric assay for tryptophan hydroxylase activity based on the different spectral characteristics of tryptophan and 5- hydroxytryptophan 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 reaction mix at 300 nm. The rate of increase of the emission signal was found to be directly proportional to the enzyme concentration. Inner filter effects due to quinonoid dihydropterin accumulation were eliminated by the inclusion of a thiol reductant. Activity measured using this assay method was found to be the same as that determined by established discontinuous HPLC assay methods. The application of the assay to routine activity measurements and to steady-state determinations with the substrates tryptophan and tetrahydropterin is described.

Original languageEnglish (US)
Pages (from-to)148-152
Number of pages5
JournalAnalytical Biochemistry
Volume266
Issue number1
DOIs
StatePublished - Jan 1 1999

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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