Purification of hydroperoxide lyase from pea seeds

Hydroperoxide lyase (HPL) development in pea seeds/seedlings was studied during germination. HPL activity in seedlings appeared after 2 days, increased to a maximum at 4 days (3.7 μmol min−1 ml−1) and decreased slowly after 8 days (2.56 μmol min−1 ml−1). Protein content decreased over 8 days from a maximum of 12.3 to 2.39 mg/ml. This resulted in an approximate 100-fold increase in specific activity of HPL from 0.01 μmol min−1 mg−1 (day 2) to 1.07 μmol min−1 mg−1 (day 8). The pI of solubilised pea seedling HPL was found to be 5.23±0.23. HPL was extracted from pea seedlings and purified by centrifugation, solubilisation with detergent, ion-exchange chromatography and hydroxylapatite chromatography. 9-Hydroperoxy-linoleic acid and 13-hydroperoxy-linoleic acid lysing activities were purified similiarly12-fold and 10-fold, respectively. The purified HPL preparation consisted of a single major band, following SDS-electrophoresis, with a molecular weight of about 55 000Da; pH 6.5 was optimum for the lysis of both 9-hydroperoxy-linoleic acid and 13-hydroperoxy-linoleic acid substrates. The enzyme was relatively unstable and lost two thirds of the original activity after 1 week at 4°C. The substrate specificity of the hydroxylapatite-purified HPL was determined by examining the activities of the HPL with various PUFA hydroperoxides. 13-Hydroperoxy-linolenic acid was 1.8, 0.8 and 1.5 times more readily lysed than 9-hydroperoxy-linoleic acid, 9-hydroperoxy-linolenic acid and 13-hydroperoxy-linoleic acid, respectively. Apparent Km values for 9-hydroperoxy-linoleic acid, 9-hydroperoxy-linolenic acid, 13-hydroperoxy-linoleic acid and 13-hydroperoxy-linolenic acid were 4.5, 14.2, 11.1 and 6.2 μM, respectively. Corresponding Vmax values were 0.12, 0.12, 0.22 and 0.26 μmol min−1. The Vmaxapp/Kmapp values for 9-hydroperoxy-linoleic acid, 9-hydroperoxy-linolenic acid, 13-hydroperoxy-linoleic acid and 13-hydroperoxy-linolenic acid were 0.03, 0.01, 0.02 and 0.04, respectively.