Characterization and inhibition studies of hemolymph phenoloxidase from Dociostaurus maroccanus

Abstract

Phenoloxidase (PO) plays a key role in insects’ immunological and physiological processes. In this study, PO from hemolymph of Dociostaurus maroccanus, which is an important pest of crops in the world, is purified in three steps: ammonium sulfate precipitation, gel-filtration and ion-exchange chromatography. The biochemical properties of PO were characterized using l-dihydroxyphenylalanine (l-DOPA) as a substrate. The optimal pH and temperature of the purified enzyme were 7 and 35 °C, respectively. A single band with a molecular weight of 73 kDa was appeared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assay. The effects of ions and chemical materials showed that enzyme activity was significantly inhibited by Zn2+. IC50 values of inhibitors on PO activity were determined as 13.14, 32.41 and 48.2 μM for 4-hexylresorcinol, kojic acid and quercetin, respectively. The inhibitory potencies (i.e. I50 of each inhibitor/I50 of 4-hexylresorcinol) of 4-kojic acid and quercetin were determined to be 2.44 and 3.66, respectively. The inhibition type of PO by 4-hexylresorcinol was obtained as competitive inhibitor, kojic acid and quercetin were found to be mixed inhibitors. Biochemical characterization of PO, as one of the most important enzymes in immune system of insect, is essential for achieving new and safe methods for pest control.