title: Purification and characterization of phenoloxidase from brine shrimp Artemia sinica.
abstract: Phenoloxidase from Artemia sinica (AsPO) was purified by Superdex 200 gel-filtration and Q Sepharose fast flow ion-exchange chromatography, and its properties were characterized biochemically and enzymatically by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. Results showed that AsPO was isolated as a monomeric protein of 125.5 kDa in molecular mass. The optimal pH value and temperature are 7.0 and 50°C, respectively, for its PO activity. The AsPO had an apparent K(m) value of 4.2 mM on L-DOPA, and 10.9 mM on catechol, respectively. Oxidase inhibitor on PO activity showed that the AsPO was extremely sensitive to ascorbic acid, sodium sulfite, and citric acid; and was very sensitive to cysteine, benzoic acid, and 1-phenyl-2-thiourea. Combined with its specific enzyme activity on L-DOPA and catechol, it can be concluded that AsPO is most probably a typical catechol-type O-diphenoloxidase. Its PO activity was also sensitive to metal ions and chelators, and 20 mM DETC-inhibited PO activity was obviously recovered by 15 mM Cu(2+), indicating that AsPO is most probably a copper-containing metalloenzyme. All these data about specific substrate, sensitivity to oxidase inhibitor metal ions and chelators indicate that the AsPO has the properties of a catechol-type copper-containing O-diphenoloxidase that functions as a vital humoral factor in host defense via melaninization as in other Crustaceans.
authors.affiliation: Department of Marine Biology, College of Marine Life Sciences, Ocean University of China, Qingdao. tjfan@ouc.edu.cn
authors.forename: Tingjun
authors.initials: T
authors.lastname: Fan
authors.forename: Zhao
authors.initials: Z
authors.lastname: Jing
authors.forename: Xianyuan
authors.initials: X
authors.lastname: Fan
authors.forename: Miaomiao
authors.initials: M
authors.lastname: Yu
authors.forename: Guojian
authors.initials: G
authors.lastname: Jiang
mesh_terms: D000818:Animals; D001156:Artemia / Q000201:enzymology*; D001205:Ascorbic Acid / Q000494:pharmacology; D019817:Benzoic Acid / Q000494:pharmacology; D055162:Biocatalysis / Q000187:drug effects; D004156:Catechol Oxidase / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D019343:Citric Acid / Q000494:pharmacology; D003300:Copper / Q000737:chemistry; D003545:Cysteine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010670:Phenylthiourea / Q000494:pharmacology; D013379:Substrate Specificity; D013447:Sulfites / Q000494:pharmacology; D013696:Temperature
chemical_list: D002396:Catechols; D004791:Enzyme Inhibitors; D013447:Sulfites; D019343:Citric Acid; D007980:Levodopa; D010670:Phenylthiourea; D003300:Copper; D019817:Benzoic Acid; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase; D003545:Cysteine; C034221:catechol; D001205:Ascorbic Acid; C025026:sodium sulfite