Photogeneration of Superoxide Anion in Serum of Bovine Milk and in Model Systems Containing Riboflavin and Amino Acids

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Abstract
Superoxide anion was generated in serum of bovine milk following illumination with fluorescent light. This was shown by suppression of nitro blue tetrazolium reduction by superoxide dismutase. The production of superoxide anion was not evident in dialyzed milk serum, but addition of 3.2 .mu.M of riboflavin to the dialyzed serum restored its production to about 50% that of the undialyzed serum. Apparently serum components of low and high molecular weight served as oxidizable substrates to photoreduce riboflavin which in turn reduced oxygen to superoxide anion. A model system equivalent in pH and riboflavin concentration to milk was used to evaluate low molecular weight compounds in milk serum for their ability to photogenerate superoxide anion. Of the compounds tested, only orotic acid formed any of it when tested at concentrations typical of milk. Among 23 amino acids evaluated, only cysteine, methionine, histidine, tyrosine and tryptophan supported superoxide anion photogeneration. The initial rate of photogeneration for the cysteine-riboflavin system was 4 times greater than for the other amino acids sensitive to oxidation. Blocking the carboxyl group of methionine reduced production of superoxide anion by more than 50% whereas blocking the amino group had a negligible effect. Changing the pH of model methionine-riboflavin systems from 6.7 to 4.5 drastically decreased the production of superoxide anion. FMN, FAD and xanthine oxidase in the presence of methionine were 77, 26 and 4% as effective as riboflavin, on an equivalent riboflavin basis, in generating superoxide anion. Native RNase yielded small quantities of it upon illumination in the presence of riboflavin, but generation increased about 15 fold upon reduction of the RNase. Reoxidation of the reduced RNase resulted in a return of superoxide anion production similar to that of the native RNase.