Measurement of xanthine oxidase in biological tissues

L. S. Terada, J. A. Leff, J. E. Repine

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

This chapter discusses the role of xanthine oxidase in biological tissues. Xanthine oxidase appears to exist in nature in two distinct functional forms, an oxidase (XO) and a dehydrogenase (XD). In addition, xanthine oxidase has a liberal substrate specificity that includes alcohols, aldehydes, and other purines, such as hypoxanthine. The conventional method for determining XO activity involves following the rate of uric acid (UA) formation from xanthine spectrophotometrically at 295 nm in the presence or absence of NAD+. Although this reaction is physiologically relevant and the substrate (xanthine) is highly specific for XO and XD, this procedure is limited by its lack of sensitivity. In addition, other compounds that absorb light at 295 nm may reduce the specificity of the assay. An improved procedure involves incubation of the enzyme with the same substrates, assaying for UA levels by high-performance liquid chromatography (HPLC). This allows for a longer incubation, and hence increasing sensitivity. HPLC analysis also permits the specific determination of UA formation, as NAD+, xanthine, ethylenediaminetetraacetic acid (EDTA), and perhaps other molecules relevant to the assay system all absorb light at 295 nm. The reaction pH of 7.8 is higher than physiologic intracellular pH to increase the sensitivity of the assay, as the pH optima of both XO and XD are 8.0 to 9.0.

Original languageEnglish (US)
Pages (from-to)651-656
Number of pages6
JournalMethods in Enzymology
Volume186
Issue numberC
DOIs
StatePublished - Jan 1 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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