[Purification and partial biochemical characterization of phenoloxidase from Penaeus chinensis].
Using L-DOPA as a probe, phenoloxidase (PO) from Penaeus chinensis hemolymph was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular weight and enzymatic properties in this study. It was found that prophenoloxidase (proPO) isolated as monomeric protein had a molecular weight of 87.5 kD, and a 77 kD phenoloxidase molecule was often contained in preparations. The 87.5 kD proPO had a very low enzymatic activity on 0.02 mmol/L L-DOPA, whereas the 77 kD PO had a very high enzymatic activity on L-DOPA. Enzymatic activity of PO against L-DOPA was optimal at pH 6.0, temperature of 40 degrees, and with an apparent K(m) value of 1.99 mmol/L. The PO activity was extremely sensitive to ascorbic acid, cysteine and dithiothreitol, very sensitive to thio urea, however not sensitive to sodium sulfite, citric acid and benzoic acid, confirmed that it was a phenoloxidase, combined with its specific oxidase activity on substrate of monophenol and L-DOPA, indicate that it was a tyrosinase type phenoloxidase. This enzyme is very sensitive to EDTA and metal ions, its activity is strongly enhanced by Mg(2+) and strongly inhibited by Cu(2+), which indicates that this PO is probably a kind of metalloenzyme.
Fan
Ting-Jun
TJ
Department of Marine Biology, College of Marine Life Sciences, Ocean University of Qingdao, Qingdao 266003, China. tjfan@ouqd.edu.cn
Wang
Xiao-Feng
XF
D000180:Acrylic Resins; D000818:Animals; D002847:Chromatography, Agarose / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000032:analysis* / Q000037:antagonists & inhibitors; D033561:Penaeidae / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate
D000180:Acrylic Resins; D004791:Enzyme Inhibitors; D012967:Sodium Dodecyl Sulfate; D014442:Monophenol Monooxygenase