Purification and characterization of phenoloxidase from immunized haemolymph of Schistocerca gregaria

Abstract

Phenoloxidase (PO) is a key factor in insect immunity. On invasion of microorganisms and pathogens, prophenoloxidase (PPO) changes to its active form, PO. The present study has been conducted to purify and characterize the PO from the haemolymph of desert locust, Schistocerca gregaria (Forskal) following activation of immune system by invasion of bacteria, Bacillus thuringiensis kurstaki (Bt). PO is purified by a combination of ammonium sulfate precipitation, blue sepharose CL-6B and phenyl sepharose CL-4B chromatography yielded a 209.97-fold purity and 54.75% recovery of activity. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) reveals that the molecular weight of the purified PO is 70.154 kDa. The purified PO is characterized in terms of its biochemical and enzymatic properties by using L-DOPA as a specific substrate. Ca²⁺ and Cu²⁺ significantly stimulated PO activity when compared with other metals. The PO reaction was strongly inhibited by phenylthiourea and thiourea, moderately inhibited by ethylene diamine tetractic acid (EDTA) and poorly inhibited by ethylene glycol tetraacetic acid (EGTA) and diethyl dithiocarbamate (DTC). Inhibition of PO showed excellent recovery ability by addition of Ca²⁺ on EGTA-inhibited enzyme. Therefore, PO is most probably a kind of tyrosinase-type Ca²⁺-containing metalloenzyme. The content of Ca²⁺ is higher than other trace metal elements. The reactive intermediates yielded by PO with its specific substrate L-DOPA had a broad-spectrum bactericidal activity against Gram +ve bacteria (Bacillus cereus and Staphylococcus aureus) with a greater degree more than Gram-ve bacteria (Escherichia coli and Pseudomonas aeruginosa). From the present study, PO from S. gregaria is most probably a tyrosinasetype calcium-containing mono-phenoloxidase, which functions not only as a catalytic enzyme in melanin production in locusts, but perhaps also as a humoral factor in host defense via melaninization as in other insects.

Key words: Schistocerca gregaria, phenoloxidase, purification.