Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: structural domains in the polypeptide chain.

Abstract

alpha-Hemocyanin from the Roman snail Helix pomatia is composed of polypeptide chains with a molecular weight of 360000 +/- 30000. The cylindrically shaped hemocyanin molecule contains 20 of these large chains. The polypeptide chain has been split into components with molecular weights of: 210000, 154000, 147000, 112000, 120000, 98000, 55000, and 50000, by gentle proteolysis with enzymes of different specificities. Most of the fragments have molecular weights which are about 50000 or a multiple of 50000. Departure from these values, as found in the 112000 and 120000 fragments, is probably caused by the high carbohydrates content of these components. A mixture of these fragments has the same oxygen binding properties as the nondigested protein. Subtilisin converts the hemocyanin polypeptide chain, under appropriate conditions, almost completely into fragments of 50000 and 55000 daltons with conservation of the oxygen binding properties of the nondigested protein. We conclude from these studies that the polypeptide chain of Helix pomatia alpha-hemocyanin is folded into about seven compact tertiary structures, which are covalently interconnected. This chain of structural domains has been visualized. (Siezen and Van Bruggen (1974), J. Mol. Biol. 90, 77-89) by electron microscopy, which shows 1/20 hemocyanin molecules to be flexible structures consisting of 7-8 apparently spherical units of 55-60 A diameter.