Purification And Partial Characterization Of Phenoloxidase From Gray Octopus Ocellatus

PO (Phenoloxidase), a widely distributed enzyme, is known to be a key enzyme in the melanogenic pathway that catalyzes the initial rate-determining reaction, the oxygenation of monophenols to o-diphenols (monophenolase activity), as well as the oxidation of o-diphenols to corresponding o-quinones (o-diphenolase activity),then o-quinones form melanin. PO has important role of immune defense reaction in invertebrate (arthropod and mollusk), and principally join the creation of skin pigment in vertebrate. In cephalopod, PO is a key enzyme in the melanogenesis creation in ink sac. Until now, there is no report about the PO form Octopus ocellatus.Octopus ocellatus belongs to Mollusca,Cephalopoda, Octopoda, Octopodidae, and is of important ecnomic value. Purification and characterization of phenoloxidase from Octopus ocellatus, not only have significance in understanding its biological functions for melanogenesis,but also help us to cognize its evolutional status.PO from Octopus ocellatus ink was purified by gel-filtration and ion-exchange chromatography, and partial characterized the biochemical properties and enzymatic properties. It was found that PO of Octopus ocellatus is a protein with a molecular weight of 153.8 kDa, composed of two subunits with 75.6 kDa and 73.0 kDa, that probably linked by a disulfide bond. Enzymatic activity of PO against L-DOPA was optimal at pH 7.0 and temperature of 40 oC, respectively. And the Km value of PO was 3.1 mmol/L on L-DOPA, and 6.3 mmol/L on catechol, respectively. It showed that L-DOPA had a higher affinity with PO than catechol. PO activity was extremely sensitive to sodium sulfite and catechol oxidase specific inhibitor 1-phenyl-2-thiourea, sensitive to ascorbic acid, thio urea, citric acid and benzoic acid. Combined with the result that Octopus ocellatus PO's oxidation of L-DOPA and catechol, but failure with tyrosine and hydroquinon, it indicated that Octopus ocellatus PO was possible a kind of catechol oxidase. The PO activity was very sensitive to...