Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae).
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 M(r) determined 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 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees 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.
Chareonthiphakorn
Nopphakaew
N
Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat-Yai 90112, Thailand.
Wititsuwannakul
Dhirayos
D
Golan-Goldhirsh
Avi
A
Wititsuwannakul
Rapepun
R
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D028482:Hevea / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007840:Latex / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry / Q000302:isolation & purification*; D012408:Rubber / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity; D013696:Temperature
D007840:Latex; D012408:Rubber; D016660:NAD(P)H Dehydrogenase (Quinone)