PURIFICATION AND CHARACTERIZATION OF PROPHENOLOXIDASE FROM THE HEMOLYMPH OF LOCUSTA-MIGRATORIA

Prophenoloxidase (proPO) was purified from plasma of the locust, Locus ta migratoria. This was achieved in three steps (gel filtration on S30 0, anion exchange on QMA Memsep, and affinity chromatography on blue T risacryl) without the use of anticoagulant buffer or inhibitors. The n ative protein had an apparent molecular mass of 250 kDa as determined by gel filtration and was likely composed of three non-covalently asso ciated subunits of 81 kDa. Its amino acid composition was found to be very similar to that of Bombyx mori proPO. Purified locust proPO could be converted into phenoloxidase (PO) by alpha-chymotrypsin. Using L-d opa as substrate, K-m and V-max were determined to be 1.5 mM and 5 mu M/s, respectively. (C) 1996 Wiley-Liss, Inc.