THE PROPHENOLOXIDASE FROM THE WAX MOTH GALLERIA-MELLONELLA - PURIFICATION AND CHARACTERIZATION OF THE PROENZYME

A prophenoloxidase (PPO) was purified from the hemolymph of the larvae of Galleria mellonella. A 135-fold purification of the proenzyme with 25% yield was achieved by a combination of different chromatographic methods. An alternative micropreparation of pure PPO by a novel method for native electrophoresis in polyacrylamide gel is also described. T he molecular mass of the native PPO was estimated to be 300 kDa by the pore-limit gradient electrophoresis in polyacrylamide gel, In the pre sence of sodium dodecyl sulphate, two closely migrating subunits of 80 and 83 kDa were detected under non-reducing conditions, The PPO was s hown to be a glycoprotein and its isoelectric point was 6.2. The amino -acid composition of the purified protein was similar to the PPO from Bombyx mori. The monospecific antibody raised against the purified PPO crossreacted with the (pro)phenoloxidase in hemolymph of Manduca sext a, The activation of the PPO with chymotrypsin was investigated and tw o proteins of 67 and 50 kDa were found to be products of the proteolyt ic cleavage. The N-terminus of the G, mellonella PPO was blocked, but eleven partial internal sequences were determined after fragmentation of the purified PPO with trypsin. Three of these peptides exhibited si gnificant homology with highly conserved sequences found in arthopod h emocyanins and insect storage proteins, which indicates that the PPO b elongs to this family,