Purification and characterization of a phenoloxidase in the hemocytes of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae): effects of insect growth regulators and endogenous inhibitors.

A phenoloxidase was extracted and purified from hemocytes of Ephestia kuehniella by using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose fast flow chromatographies. At the final stage of purification, a protein was purified by molecular mass of 78.5 kDa, specific activity of 1.17 U/mg protein, recovery of 20.48% and purification fold of 16.71. The purified PO showed the highest activity at pH 4-5 and temperatures of 35-40 °C. Na(+), K(+), Mn(+), Zn(2+) and Mg(2+) decreased activity of the purified PO but Ca(2+) and Cu(2+) increased the enzymatic activity. EDTA (General chelating agent), DTC (Copper chelating agent) and EGTA (Calcium chelating agent) significantly decreased PO activity but TTHA (Magnesium chelating agent) showed no statistically significant effects. Kinetic parameters of the purified enzyme showed the highest Vmax when L-DOPA was used as substrate but no significant differences were observed in case of Km for used L-DOPA, pyrocatechol and hydroquinone. In vitro inhibition of the purified PO by using two insect growth regulators, Hexaflumuron and Pyriproxyfen, revealed IC50 of 96.41 and 38.59 µg/ml for these compounds, respectively. Kinetic studies using different concentrations of L-DOPA and IC50 concentrations of the two IGRs revealed the increase of Km value versus control and competitive inhibition. Finally, column chromatography of hemolymph revealed peak III showing endogenous inhibitors of phenoloxidase by molecular weight of 27.3 that showed competitive inhibition on the PO.