Purification and characterization of phenoloxidase from crab Charybdis japonica.
Phenoloxidase (PO) from hemolymph of Charybdis japonica was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its molecular mass and enzymatic properties by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. It was found that prophenoloxidase (proPO), isolated as a monomeric protein, had a molecular mass of 69.5 kDa, and a 64.5 kDa PO molecule was often contained in preparations. The PO activity showed optimal pH of 6.0, optimal temperature of 40 degrees C, and an apparent Km value of 3.41 on L-DOPA, and 7.97 on catechol. PO activity was extremely sensitive to sodium sulfite and 1-phenyl-2-thiourea, and very sensitive to thiourea and benzoic acid. Based on its inhibition characteristics and the substrate affinity, this PO was classified as a kind of o-diphenoloxidase. The PO activity was also strongly inhibited by Zn(2+), Mg(2+), ethylenediaminetetraacetic acid (EDTA) and diethyldithiocarbamate (DETC). The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu(2+) on DETC-inhibited PO activity, indicate that Charybdis PO is most probably a copper-containing metalloenzyme.
Liu
Guangxing
G
Department of Environment Ecology, Division of Life Science and Technology, Ocean University of China, Qingdao 266003, PR China.
Yang
Lingling
L
Fan
Tingjun
T
Cong
Rishan
R
Tang
Zhihong
Z
Sun
Wenjie
W
Meng
Xianghong
X
Zhu
Liyan
L
D000818:Animals; D019817:Benzoic Acid / Q000378:metabolism; D003386:Brachyura / Q000201:enzymology*; D002845:Chromatography; D004492:Edetic Acid / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D019216:Metals, Heavy / Q000633:toxicity; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013447:Sulfites / Q000378:metabolism; D013859:Thiocarbamates / Q000633:toxicity; D013890:Thiourea / Q000378:metabolism
D019216:Metals, Heavy; D013447:Sulfites; D013859:Thiocarbamates; D007980:Levodopa; D019817:Benzoic Acid; D004492:Edetic Acid; D014442:Monophenol Monooxygenase; D013890:Thiourea; C025026:sodium sulfite