Hemocyanins in Spiders, XIV. Subunit Composition of Dissociation Intermediates and its Bearing on Quaternary Structureof EurypelmaHemocyanin

Abstract

The 37S hemocyanin (24 subunits of 7 types) isolated from the tarantula, E. 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 contained 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 a constituent hexamer of the native (4 .times. 6) hemocyanin, the 16-mer by losing 1 further copy each of e, f and g. The dodecamer is formed by cleavage with 2-mercaptoethanol and represents a 1/2 molecule. The octamer could not be clearly analyzed but probably contains 1 copy of chain d more compared to the heptamer. The heptamer represents a 1/4 hemocyanin with 1 additional polypeptide chain sticking out of the hexameric structure. This can be either subunit b or c of the heterodimer bc. Only 1 type of hexamer was obtained after cleavage of the 37S hemocyanin with 4M urea, containing 1 copy each of chains a, b, d, e, f and g. The 37S hemocyanin is composed of 2 identical dodecameric halves linked by dimerization of subunit f. Each 1/2 molecule is constituted by 2 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 heterodimer bc. The b-hexamer appears to be rather stable, the c-hexamer unstable. The relative positions of some of the subunits within the native oligomer are discussed.