Purification of hydroperoxide lyase from pea seeds

Hydroperoxide lyase (HPL) development in pea seeds/seedlings was studied du ring germination. HPL activity in seedlings appeared after 2 days, increase d to a maximum at 3 days (3.7 mu mol min(-1) ml(-1)) and decreased slowly a fter 8 days (2.56 mu mol 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 mu mol min(-1) mg(- 1) (day 2) to 1.07 mu mol min(-1) mg-' (day 8). The pI of solubilised pea s eedling HPL was found to be 5.23+/-0.23. HPL was extracted from pea seedlin gs and purified by centrifugation, solubilisation with detergent, ion-excha nge chromatography and hydroxylapatite chromatography. 9-Hydroperoxy-linole ic acid and 13-hydroperoxy-linoleic acid lysing activities were purified si miliarly 12-fold and 10-fold, respectively. The purified HPL preparation co nsisted of a single major band, following SDS-electrophoresis, with a molec ular weight of about 55 000Da; pH 6.5 was optimum for the lysis of both 9-h ydroperoxy-linoleic acid and 13-hydroperoxy-linoleic acid substrates. The e nzyme was relatively unstable and lost two thirds of the original activity after 1 week at 4 degrees C. The substrate specificity of the hydroxylapati te-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-hydropero xy-linolenic acid and 13-hydroperoxy-linoleic acid, respectively. Apparent K-m values for 9-hydroperoxy-linoleic acid, 9-hydroperoxy-linolenic acid, 1 3-hydroperoxy-linoleic acid and 13-hydroperoxy-linolenic acid were 4.5, 14 .2, 11.1 and 6.2 mu M, respectively. Corresponding V-max values were 0.12, 0.12, 0.22 and 0.26 mu mol min(-1). The V-max(app)/K-m(app) values for 9-hy droperoxy-linoleic acid, 9-hydroperoxy-linolenic acid, 13-hydroperoxy-linol eic acid and 13-hydroperoxy-linolenic acid were 0.03, 0.01, 0.02 and 0.04, respectively. (C) 2000 Published by Elsevier Science Ltd.