Fluorometric oxidase assays: Pitfalls caused by action of ultraviolet light on lipids

Abstract A number of fluorometric assays of hydrogen peroxide-producing oxidases are based on the formation of highly fluorescent products from homovanillic acid or related compounds by horseradish peroxidase. We report the observation that under continuous uv illumination at the wavelengths used for excitation in these methods, brain or muscle homogenates produce fluorescence increases in the absence of any exogenous enzyme substrate; when uv light is excluded, such increases are negligible. Arachidonic and linolenic acids also produce this effect. For this reason, measurements of H 2 O 2 based on this principle are valid only if this nonspecific effect has been excluded, and should preferably be carried out as end-point rather than continuous assays. It is believed that the effect of uv light on the reaction is due to formation of H 2 O 2 and/or oxygen free radicals, and polyunsaturated lipids appear to be involved as intermediates. Thus, the homovanillic acid-horseradish peroxidase system may prove useful in investigations of the effect of uv on the production of oxygen free radicals and lipid peroxidation.