Phenoloxidase From Gastrolina Depressa And Its Inhibitors

Phenoloxidase (PO, EC.1.14.18.1), or called tyrosinase or catechol oxidase, include monophenol and polyphenoloxidase. This enzymes have been classified into three groups based on their substrate specificity: monophenol oxidase (EC.1.14.18.1), catechol oxidase (EC.1.10.3.1) and laccase (EC.1.10.3.2). The phenoloxidase which localized in melanocytes is distributed in animals, plants, microorganisms, insect and man. It is one of the key enzymes in the development process of insects, the enzyme possesses an important function in metamorphism developing and immunity system. Currently, many studies focused on this field in order to screen, design and synthesis PO inhibitors for the importance theory of PO inhibitors and its bright future.In the present paper, the kinetic properties of phenoloxidase from adult Gastrolina depressa one kind of Coleoptera insect, were determined after the enzyme was partially purified by different staturated (NH₄)₂SO₄. Effects of some effectors on the activity of PO were determined, using catechol as substrate. The inhibitory effects on the PO from G. depressa activity by benzoic acid, citric acid, corbic acid and cysteine were determined, and the mechanism of the four inhibitors were discussed also. The results could be summarized as follows:1. Properties of the phenoloxidase (PO) from adult of G. depressa Baly purified by (NH4)2SO4 were determined. The optimal pH and temperature of the enzyme for the oxidation of catechol were determined to be at pH 7.5 and at 40℃, respectively. The kinetic parameters for the oxidation of L-DOPA and catechol by the PO were 15.01 mmol·L^-1 and 9.17 mmol·L^-1.2. Studies on the induction of some detergents and trypsin on the PO activity. The results showed that TritonX-100, SDS and trypsin all enhanced the activity of this PO.3. Studies on the effects of some metal ions on the PO activity. The results showed that K⁺ and Na⁺ had no any influence on the enzyme activity. Mg²⁺, Ba²⁺ activated the PO activity at low concentration, however, inhibited activity at higher concentration. Zn²⁺, Cu²⁺ and Fe³⁺ all exhibited inhibition effects and the IC50 were 0.09 mmol·L^-1, 0.125 mmol·L^-1 and 0.048 mmol·L^-1, respectively.4. Studies on the effects of some organic solvents on the activity of PO. The results showed that these solvents had inhibitory effects on the enzyme activity. The IC50 were 1.152 mmol·L^-1, 0.753 mmol·L^-1, 1.06 mmol·L^-1 and 1.25mmol·L^-1, by methanol, glycerol, aether and acetone. The ethanol showed slight inhibitory effects, which can inhibite most 36% activity. The inhibitory effects of dichloromethane showed ruleless.5. Studies on the inhibitory effects by benzoic acid, citric acid, corbic acid and cysteine on the activity of PO. The results showed that benzoic acid and citric acid exhibited noncompetitive reversible inhibition and the inhibitory constants (Ki) were determined to be 0.0646 mmol·L^-1 and 0.081 mmol·L^-1, respectively. However, cysteine and corbic acid exhibited reversible competitive inhibition and the inhibitory constants (Ki) were determined to be 0.40 mmol·L^-1 and 2.22 mmol·L^-1, respectively.