Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium

Mauro Maccarrone, Antonello Rossi, Gabriele D'Andrea, Gianfranco Amicosante, Luciana Avigliano

Research output: Contribution to journalArticlepeer-review

Abstract

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44, 276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 m hydrogen peroxide. Ascorbate (25 μm) or riboflavin (500 μm) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure.

Original languageEnglish
Pages (from-to)101-104
Number of pages4
JournalAnalytical Biochemistry
Volume188
Issue number1
DOIs
Publication statusPublished - 1990

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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