Purification and Characterization of Phenoloxidase from the Haemolymph of Desert Locust, Schistocerca gregaria (Forskal) Following Activation of Immune System

Abstract

The present study has been conducted to purify and characterize the phenoloxidase (PO) from the haemolymph of desert locust, Schistocerca gregaria (Forskal) following activation of immune system by invasion of bacteria, Bacillus thuringiensis kurstaki (Bt). PO purified by 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. SDS-PAGE reveals that the molecular weight of the purified PO is 70.154 kDa. In addition, rabbit antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting analysis. Twenty types of amino acids have been detected in the purified PO by High-performance liquid chromatography (HPLC). The amino acid composition was very similar to that of Locusta migratoria prophenoloxidase (PPO). The N-terminal amino acid sequence was determined by automatic protein sequencer, and 291 amino acid residues have been detected. Comparison of PO sequence to sequence databases using Basic Local Alignment Search Tool (BLAST) revealed high percentage of identity to members of order Orthoptera; 82% identity with PPO 2 [Locusta migratoria] and