Limited proteolysis of the 94 000-dalton subunit of Panulirus interruptus hemocyanin; the carbohydrate attachment site.

Abstract

Limited proteolysis of the native monomeric 94-kDa subunit of Panulirus interruptus hemocyanin by trypsin, plasmin and subtilisin produces an 18-kDa fragment, a 71-kDa fragment and a small glycopeptide. In the plasmin digest a 23-kDa precursor of the 18-kDa fragment has been observed. Automatic Edman degradations demonstrated that the 18-kDa fragments have their origin at the N terminus of the 94-kDa subunit and incubations with carboxypeptidase A showed that the 71-kDa fragments originate from the C terminus. The glycopeptide is situated in between. The amino acid sequence of the glycopeptide has been determined. Its carbohydrate content accounts for the total carbohydrate of the 94-kDa subunit. All three proteases cleave the subunit at two positions within a very restricted area of the polypeptide chain, which indicates the presence of an exposed loop, carrying the carbohydrate chain, in the corresponding part the native molecule.