Continuous Spectrophotometric Assay and Properties of Ascorbic Acid Oxidising Factors in Wheat

A continuous spectrophotometric assay was developed to measure ascorbic acid oxidation in crude Na₂SO₄ extracts of flour. The rate of ascorbic acid oxidation in flour extracts measured using this method was similar to the rate in flour-water suspensions and 2–4 fold less than the rate in dough measured using an indophenol-xylene extraction method. Flour extracts appeared to contain two ascorbic acid oxidising factors; one with optimal activity at pH 6·3 and 30 °C and the other with optimal activity at pH 10 and 40 °C. The pH 6·3 factor had properties similar to those of ascorbate oxidase (EC 1·10·3·3) in its pH and temperature stability, strong inhibition by NaN₃, KCN and diethyldithiocarbamate, inactivation by proteases, and greater stereospecificity towards -ascorbic acid than -isoascorbic acid. The pH 6·3 factor was most concentrated in the pollard milling fraction of wheat and was lowest in flour. The pH 10 factor had several properties indicating non-enzymic oxidation of ascorbic acid; it was not inactivated by proteases, it was inhibited poorly or not at all by the above ascorbate oxidase inhibitors, and it had low specificity for stereoisomers of ascorbic acid.