Hemocyanins in Spiders, IV. Subunit Heterogeneity ofEurypelma (Dugesiella)Hemocyanin, and Separation of Polypeptide Chains

Abstract

The hemocyanin of the North American tarantula E. californicum (D. californica) is dissociated at pH 9.6 into monomers (Mr [molecular ratio] about 70,000) and dimers (Mr about 140,000), which were separated by gel filtration. The monomer peak was resolved by preparative polyacrylamide gel electrophoresis and yielded 4 protein bands, 3 of which (1, 3 and 4M) were apparently homogeneous. Band 2 contained 2 sub-fractions (2I and 2II). The dimer peak contained 2 dimers (bands 4D and 5). Upon treatment with 4 mM cysteine the dimer band 5 was dissociated, yielding only 1 type of monomer identical with band 3. The other dimer which was only partially dissociated by 10 mM EDTA, was most probably a heterodimer, 1 component being electrophoretically indistinguishable from band 2II. After treatment of the native hemocyanin with sodium dodecylsulfate [SDS] and analysis in gradient gel slabs, 6 polypeptide chains were observed (a-f). They corresponded to the products of alkaline dissociation: band 1 = e, band 2I = a, band 2II = c, band 3 = f, band 4M = d, band 4D = b plus c, band 5 = f. The MW determined by SDS gel electrophoresis in gradient gels, and by sedimentation equilibrium analysis ranged between 67,000-76,000. The sedimentation coefficients were between 4.4-4.7 S for the monomers and 6.6-6.7 for the dimers. The isoelectric points ranged from pH 4.5 to pH 5.4. The findings were discussed with respect to the limitations of MW determination by conventional SDS gel electrophoresis, to the structure of the hemocyanin oligomers and to possible biological significance.