Hemocyanins in spiders: XI. The quaternary structure of Cupiennius hemocyanin

Abstract

The hemocyanin of the lycosid spider Cupiennius salei was separated into its hexameric (16 S) and dodecameric (24 S) components, and analyzed quantitatively. The reassociation and topologic distribution of the subunits were studied. There are two types of subunits. One is monomeric (5 S) and consists of 5 electrophoretically distinct bands which are, however, immunologically identical. The other is a disulphide bridged dimer (7 S) which yields 2 components upon electrophoresis or immunoelectrophoresis. The significance of this heterogeneity was not studied. The dimer is antigenically deficient with respect to the monomer. Whereas the 16 S hemocyanin is composed of six monomers, 24 S hemocyanin contains 10 monomers and 1 dimer. Alkaline dissociation of 24 S hemocyanin (dodecamer) into subunits passes through a heptameric state (18 S) which is composed of 5 monomers and the dimer. In the electron microscope, 16 S-like units with a seventh polypeptide attached can be distinguished. Treatment of 24 S or 18 S hemocyanin with reducing agents to cleave the disulphide bridge leads to a second type of hexamer (16 S′) which is electrophoretically distinct from native hexamers (16 S), and composed of 5 monomers and one constituent polypeptide chain of the dimer. Upon dialysis of a monomer/dimer mixture against neutral buffer containing 40 mM calcium, 16 S, 18 S and 24 S particles are formed. The three reconstituted hemocyanins exhibit subunit compositions identical to the native hemocyanins and the 18 S component obtained during dissociation. The results suggest that the 24 S hemocyanin particle consists of two identical hexamers linked by the disulphide bridge of a dimeric subunit shared by both hexamers. © 1980, Springer-Verlag. All rights reserved.