Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: isolation of a functionally active chemically pure domain and evidence for subunit heterogeneity.

α-Hemocyanin of the vineyard snail, Helix pomatia, is a large oligomer composed of 20 subunits with a molecular weight of 360,000 ± 30,000. Limited proteolysis showed these subunits to be composed of about seven structural domains, each having one oxygen binding site (1). This paper describes the production of these structural domains by tryptic digestion of $\text{1}\text{10}$ hemocyanin molecules. The digestion pattern was followed as a function of time by examining the proteolytically obtained fragments electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels. Based on the molar ratio of each fragment present during digestion the first part of the reaction sequence for trypsinolysis could be deduced. This reaction scheme was simulated by means of an analog computer. Pseudo-first-order reaction rate constants of the various proteolytic cleavages were estimated from the computer generated time course of digestion. On the basis of these results it is postulated that Helix pomatia α-hemocyanin possesses at least two kinds of subunits which differ in their proteolytic susceptibility. These subunits occur in equimolar amounts. A functionally active domain with a molecular weight of about 50,000 has been isolated from a tryptic digest of hemocyanin subunits. This domain seems to be chemically pure, as suggested by the unique sequence of its first six amino acids, viz: Lys-Val-His-Leu-Asn-Lys.