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Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium
M Maccarrone1, A Rossi, G D'Andrea
1Department of Biomedical Sciences and Technologies and Biometrics, University of L'Aquila, Italy.
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 microM) or riboflavin (500 microM) 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.