RESUMO
Hemocyanin is a copper containing protein and its role in the immune function of phenoloxidase (PO) activity was investigated in the giant freshwater prawn Macrobrachium rosenbergii. Hemocyanin, sedimented by ultracentrifugation from the plasma appeared on polyacrylamide gel electrophoresis (PAGE 7%) on Coomassie Brilliant Blue and bathocuproine sulfonic acid stain as four copper containing proteins of molecular masses 50, 60, 114 and 325kDa. Accordingly, on diethylaminoethyl-cellulose anion exchange column hemocyanin separated into four proteins designated as MrHc1, MrHc2, MrHc3 and MrHc4 with electrophoretically (PAGE) determined molecular masses of 60, 114, 50 and 325kDa respectively. The reduction of proteins in sodium dodecyl sulphate (SDS)-PAGE revealed that MrHc1 and 3 were monomeric for 60and 50kDa respectively, MrHc2 dimeric of 56 and 58kDa subunits and MrHc4 appeared with three subunits of 74, 76 and 78kDa. The PO activity was determined in plasma, hemocyanin and the four separated hemocyanin proteins in vitro using L-3,4-dihydroxyphenylalanine (L-DOPA) at pH7.5, 25°C and appeared elicited by exogenous activators such as trypsin, SDS, cell wall components of bacteria and polysaccharide laminarin. This study clearly demonstrated hemocyanin as the major copper containing protein in the plasma of M. rosenbergii with potent PO activity.