Arrangement of functional units within the Rapana thomasiana hemocyanin subunit RtH2

For the determination of the number and linear sequential arrangement of fu nctional units (FUs) within the polypeptide chain of the Rapana hemocyanin subunit RtH2, a panel of mono-, di-, tri- and penta-FU fragments was genera ted by limited proteolysis of the purified subunit with four different enzy mes. The individual cleavage products were isolated, characterized by SDS-P AGE and N-terminally sequenced. Most of the information about the FU sequen tial arrangement within RtH2 was obtained after limited proteolysis of the subunit with plasmin. Overall correlation of the data revealed the sequenti al order of the eight FUs within the polypeptide chain of RtH2, termed RtH2 -a to RtH2-h. The sites, most sensitive to proteolytic cleavage with plasmi n, are located at the C-terminus, between the FUs ef, fg and gh. A second m ain cleavage site was observed between the FUs ed. Endoproteinase GluC hydr olyzes these sites, too, but produces exclusively a mixture of mono-, di- a nd tri-FU fragments. The most stable fragments, the trimer abe and the dime r gh, are found in all cleavage mixtures of RtH2 studied. RtH2-h is compare d with the corresponding h-FUs of the gastropodan hemocyanins of Pila leopo ldvillensis, Helix pomatia, Megathura crenulata and Haliotis tuberculata, a nd a remarkable similarity is observed between them: an increased M-r of ap proximate to 65 000 instead of approximate to 50 000, estimated for an aver age FU, suggesting that the sequence of RtH2-h is elongated by about 95 ami no acid residues at the C-terminal part of the molecule, as found for beta( c)-HpH, HtH1 and HtH2. (C) 2000 Elsevier Science B.V. All rights reserved.