Hemocyanins in Spiders, XIV. Subunit Composition of Dissociation Intermediates and its Bearing on Quaternary Structure of Eurypelma Hemocyanin

The 37 S hemocyanin (24 subunits of 7 types) isolated from the tarantula, Eurypelma californicum, was dissociated partially by various agents and the dissociation intermediates analyzed for their subunit composition by crossed immunoelectrophoresis. The subunit composition of the native hemocyanin was reexamined and the pending problem of the ratio between subunits a and g (= c2) clarified. The subunits are present in the ratio of a:b:c:d:e:f:g = 4:2:2:4:4:4:4. Breakdown products were shown to contain 19 (20?), 16, 12, 8, 7 and 6 polypeptide chains (possibly, there is also a 15-mer). The 19-mer is formed by removal of 5 monomeric subunits from one of the constituent hexamers of the native (4 x 6) hemocyanin, the 16-mer by losing one further copy each of e, f and g. The dodecamer is formed by cleavage with 2-mercaptoethanol and represents a half-molecule. The octamer could not be clearly analyzed but probably contains one copy of chain d more compared to the heptamer. The heptamer represents a one-quarter hemocyanin with one additional polypeptide chain sticking out of the hexameric structure. This can be either subunit b or c of the heterodimer bc. Only one type of hexamer was obtained after cleavage of the 37 S hemocyanin with 4M urea, containing one copy each of chains a, b, d, e, f and g. It is concluded that the 37 S hemocyanin is composed of two identical dodecameric halves linked by dimerization of subunit f, and that each half-molecule is constituted by two non-identical though similar hexamers, both encompassing a complete set of subunits a, d, e, f and g, but differing in their share of the rather stable, the 'c'-hexamer unstable. The relative positions of some of the subunits within the native oligomer are discussed.