Purification and characterization of phenoloxidase from the hemolymph of healthy and diseased Antheraea assamensis Helfer (Lepidoptera: Saturniidae): Effects of certain biological components and chemical agents on enzyme activity.

In the current study, a dimeric phenoloxidase (PO) from the hemolymph of healthy and diseased (pebrine infected) larvae of Antheraea assamensis Helfer was extracted and purified. The protein was subjected to purification using Sephacryl S-100 and CM Sepharose chromatography. The enzyme comprised of two subunits of ~76.8 and 76 kDa that showed PO activity in 6 mM l-3,4-dihydroxyphenylalanine (L-DOPA) and 8 mM catechol but not in hydroquinone. Optimum temperature for PO activity was 30°C in l-DOPA and 37°C in catechol. Optimum pH ranged from 6.8 to 7.0 in L-DOPA and 7.0-7.2 in catechol. Specific activity of the purified PO from healthy larvae was 53.9 µM/min per mg of protein per ml in L-DOPA and 50.77 µM/min per mg of protein per ml in catechol. Specific activity of PO from diseased larvae was 30.0 µM/min per mg of protein per ml in L-DOPA and 28.55 µM/min per mg of protein per ml in catechol. Purification fold was 3.27-4.21 for healthy and 2.38-2.56 for diseased fractions. The enzyme showed the Michaelis constant (Km ) of 2.46-2.85 mM for healthy and diseased fractions in L-DOPA. In catechol Km of 9.23-17.71 mM was observed. Peptidoglycan was the best activator of purified PO from both healthy and diseased fractions. Interactions between controls and activators appeared statistically significant (F = 767.5; df = 3; P < 0.0001). Na+ , K+ , and Cu2+ increased, whereas Ca2+ , Zn2+ , Mg2+ , and Co2+ decreased PO activity. The overall interactions appeared highly significant (F = 217.0; df = 27; P < 0.0001). Kojic acid, dithiothreitol, thiourea, phenylthiourea, carbendazim, N-bromosuccinimide, N,N,N',N'-tetraacetic acid, and diethyldithiocarbamate inhibited PO activity.