Purification and characterization of phenoloxidase from <italic>Octopus ocellatus</italic>

Abstract

Phenoloxidase (PO) from ink sacs of Octopus ocellatus was purified by gel-filtration and ion-exchange chromatography, and characterized in terms of its biochemical and enzymatic properties by using L-dihydroxyphenylalanine (L-DOPA) as the specific substrate. It was found that prophenoloxidase from O. ocellatus was isolated as a heterodimeric protein of 153.8 kDa, and two subunits of 75.6 and 73.0 kDa were often detected in preparations after SDS activation. The PO-like activity showed optimal pH of 7.0, optimal temperature of 408 8 8 8 8C, and an apparent K m value of 3.1 mM on L-DOPA, and 6.3 mM on catechol, respectively. The PO-like activity was extremely sensitive to 1-phenyl-2-thiourea and sodium sulfite, and very sensitive to ascorbic acid, thiourea, citric acid, and benzoic acid. Together with its specific enzyme activity on catechol and L-DOPA, it can be concluded that the Octopus PO is most probably a typical o-diphenoloxidase. The PO-like activity was also strongly inhibited by Cu 21 , Zn 21, ethylenediaminetetraacetic acid and diethyldithiocarbamate (DETC), and the DETC-inhibited PO-like activity could be perfectly restored by Cu 21 . These results indicated that Octopus PO is most probably a copper-containing metalloenzyme. All these results implied that the PO from O. ocellatus has the properties of a catechol-type copper-containing o-diphenoloxidase which functions not only as a catalytic enzyme in melanin production in ink sacs but also as a humoral factor in host defense via melaninization as in other crustaceans.

Purification and characterization of phenoloxidase from brine shrimp <italic>Artemia sinica</italic>

“…The estimated molecular mass of the purified AsPO was determined to be 125.5 kDa in both reducing and non-reducing SDS-PAGE, which indicated that only one type of active PO in brine shrimp A. sinica. The molecular mass of AsPO is much higher than that of the other Crustacean POs (60-77 kDa) [7,10,11,13 -15,17], and lower than that of heterodimeric proPOs from O. ocellatus (153.8 kDa) [11] and squid Illex argentinus tyrosinase (140.2 kDa) [25]. These results imply that AsPO is different from the other Crustacean POs both in molecular forms and molecular masses.…”

“…The reaction was stopped and each reaction mixture was measured spectrophotometrically under the same conditions as described above, respectively. To determine the optimal pH value of AsPO activity, the reaction mixture containing 20 ml of 15 mM L-DOPA and 20 ml of the purified AsPO at pH 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, was incubated for 40 min at 288C as described previously [11]. The reaction was stopped and each reaction mixture was measured spectrophotometrically under the same conditions as described above, respectively.…”

“…It has been well proved that phenoloxidase (PO), an active form of prophenoloxidase ( proPO), is a copper-containing key enzyme in the innate defense system of invertebrates [1][2][3][4][5][6][7][8][9][10][11]. PO, under stimulation of invading pathogens, can be activated by the vital cascade reaction of 'proPO activating system' in a stepwise process, which results in pathogen encapsulation by formation of melanins that is a vital melanogenic pathway involved in immune reactions of invertebrate hemocytes [1,4,5,8,12].…”

“…Crustacean and Mollusca proPOs, synthesized in blood cells, have been isolated and characterized from several species [3][4][5]7,[9][10][11]14]. It has been found that the purified Crustacean and Mollusca proPOs are monomers with a molecular mass of 70-87 kDa, while their activated forms were 60-77 kDa [7,9,10,14 -16].…”

“…However, the proPO from Octopus ocellatus has been identified as a heterodimeric protein with a molecular mass of 153.8 kDa and presents two subunits with molecular mass of 75.6 and 73.0 kDa after sodium dodecyl sulfate (SDS) activation [11]. Moreover, Crustacean and Mollusca POs have properties of either tyrosinase-type of metalloenzymes in brown shrimp Penaeus californiensis [17], shrimp Penaeus chinensis [7], and clam Ruditapes philippinarum [9], or O-diphenoloxidase-type of metalloenzymes in Charybdis japonica, [10] and O. ocellatus [11].…”