Phytochemistry, Vol. 27, No. 2, pp. 323-327, 1988. 0031 9422/88$3.00+0.00 Printed in Great Britain. © 1988 PergamonJournals Ltd. PURIFICATION AND CHARACTERIZATION OF AVOCADO LIPOXYGENASE LIONEL MARCUS, DOV PRUSKY~ and BENJAMINJACOBY3~ Department of Fruit and Vegetable Storage, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel; :~Department of Agricultural Botany, Faculty of Agriculture, Hebrew University of Jerusalem, P.O.B. 12, Rehovot, 76100, Israel. (Revised received 9 July 1987) Key Word lndex--Persea americana; Lauraceae; lipoxygenase; purification; affinity chromatography Abstract--Lipoxygenase (E.C. 1.13.1.13) from the avocado cultivar 'Fuerte' was purified to near homogeniety by affinity chromatography. The enzyme was extracted in potassium phosphate buffer at pH 7.2 in the presence of 2% Triton X-100. Triton was removed from the homogenate by adsorption on 25.0-350 mesh activated charcoal. Lipoxygenase was partially purified (seven-fold) by 66% acetone precipitation from a 20% acetone supernatant. The precipitate was dissolved in a potassium phosphate buffer at pH 7.2 and loaded on an affinity chromatography column. This single-step chromatographic purification yielded a single lipoxygenase activity peak. The total activity yield of the purification procedure was ca 65% and the degree of enrichment ca 35-fold. The M, determined by gel filtration and by electrophoresis, was 74 000. Optimum enzyme activity was found at 36 ° and pH 7.1. The energy of activation, amino acid composition, isoelectric point, kinetic parameters and inhibitory effect of epicatechin were studied. The enzyme was found to obey Michaelis-Menten kinetics. The Km of the avocado lipoxygenase for linoleate was 7.2 × 10-2 mM and the VmaxWaS432 #mol/hr/mg. Epicatechin acted as a competitive inhibitor with a Ks of 9.0 × 10-s mM. INTRODUCTION In the presence of molecular oxygen, lipoxygenase (linol- eate: O: oxidoreductase E.C. 1.13.1.13.) catalyses the oxidation of C18-unsaturated fatty acids with a cis,cis-1,4- pentadiene group to give hydroperoxide [1]. Several conventional and non-conventional methods for the isolation of this enzyme from a variety of plant and animal tissues have been described [2-7]. Affinity chromato- graphy purification was reported by Grossman et al. [8] for soybean lipoxygenase, and recently by Cohen et al. [9] for that from pea. Attention has been drawn recently to avocado lipoxygenase because of its suggested physio- logical role in the breakdown of the antifungal 1-acetoxy- 2-hydroxy-4-oxo-heneicosa-12,15-diene involved in the resistance of unripe avocado fruit to fungal infection [10-12]. The present paper describes conditions for the purification of avocado lipoxygenase by affinity chroma- tography and the catalytic properties of this enzyme. RESULTS Cold acetone (4o ) was added to the crude enzyme preparation to a concentration of 20% v/v and the precipitate was removed after 1 hr by centrifugation at 20000 g for 10 min. The supernatant was adjusted with Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 1941-E, 1987 series. tAuthor to whom correspondence should be addressed. additional acetone to 66% v/v and after 2 hr, again centrifuged at 20000 g for 10 rain. The precipitate was dissolved in 200ml of 20mM potassium phosphate buffer, pH 7.2, and the insoluble fraction separated by centrifugation. A 10 ml aliquot of the supernatant was applied to the column of cross-linked Sepharose- hexamethyl-linoleate (10 x 0.7 cm) and eluted with the same buffer at a flow rate of 18 ml/hr. The eluent was collected in 6 ml fractions. Protein from the column was monitored by absorbance at 280 nm. The elution profile of the crude enzyme preparation from the cross-linked linoleate column showed that most of the inactive protein was eluted with the buffer (Fig. 1). The lipoxygenase activity remained tightly bound to the column even after prolonged washing with the buffer. The bound enzyme was released from the linoleate column with a linear gradient of sodium chloride, 0.0-0.5 M, in the same buffer. Elution commenced when the NaCI concentration approached 0.2 M. Fractions containing lipoxygenase activity were pooled, dialysed against 20 mM potassium phosphate buffer, pH 7.2, for 24 hr, lyophilized and redissolved in 2 ml of distilled water. This solution was used as a purified enzyme source for further study. The yields, degrees of enrichment and specific activity of the avocado lipoxygenase for a typical purification procedure are summarized in Table 1. In this experiment the specific activity of the purified enzyme was 18.35 pmol O2/min/mg protein (0.3 × 10-6 Kat/mg) and the enrich- ment was 35-fold. The purity of the enzyme was evidenced by the single protein band obtained by SDS gel electro- phoresis. Cross-linking with ethylene diamine was also explored, but purification by this column was not ade- 323