Purification and characterization of phenoloxidase from the hemolymph of Hyphantria cunea (Lepidoptera: Arctiidae)

Abstract

Phenoloxidase (PO) is a key factor in insect immunity. On invasion of microorganisms and
pathogens, prophenoloxidase changes to its active form to PO. This study investigated purification biochemical properties of PO from the hemolymph of 5th instar larvae of Hyphantria cunea (Lepidoptera). The purification fold was determined as 9.67 with a recovery of 0.12 and a specific activity of 23.28 U/mg protein identified. Kinetic parameters of purified PO from the insect H. cunea were determined. The Michaelis constant (Km) and the maximal velocity (Vmax) were 4.08 and 12.98 µmol/min/mg protein, respectively. The optimal pH and temperature of the enzyme for oxidation of LDOPA were 10.0 and 35 º C, respectively. The ions Zn2+, Cu2+, K+ and Na+ significantly increased the enzyme activity and synthetic inhibitors such as diethyldithiocarbamate (DETC) significantly decreased it. Finally, it was found that purified PO had a molecular mass of 33 kDa. This study demonstrated some PO properties and its inhibitory effects demonstrating that it can be employed as useful methods for developing novel insecticides to replace traditionally used ones.