Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae)

Abstract

NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The Mrdetermined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 °C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 °C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity, Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r = 0.89, P <0,01), dry rubber (r = 0.81, P <0,01)] together with flow time (r = 0.85, P <0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones. © 2002 Elsevier Science Ltd. All rights reserved.