A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

G. J. Peters, E. Laurensse, A. Leyva, H. M. Pinedo

Research output: Contribution to journalArticle

Abstract

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80-1000 μm) both Km and Vmax values were comparable. With the HPLC assay the concentration range was extended down to 12 μm and initial rates could be determined. The apparent Km was about 12 μm. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.

Original languageEnglish (US)
Pages (from-to)32-38
Number of pages7
JournalAnalytical Biochemistry
Volume161
Issue number1
DOIs
StatePublished - Feb 15 1987
Externally publishedYes

Keywords

  • dihydroorotic acid dehydrogenase
  • enzyme assay
  • high-performance liquid chromatography
  • orotic acid
  • pyrimidine de novo
  • pyrimidine nucleotides

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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