Tachypleus tridentatus hemocyanin. Separation and characterization of monomer subunits and studies on sulfhydryl...

Hemocyanin monomers were isolated from Tachypleus tridentatus hemolymph by gel chromatography on Sephadex G-100. The isolated monomers were separated into four fractions by DEAE-Sephadex chromatography; they comprised 6 different subunits, designated as alpha-zeta chains. All of them showed the same molecular weight, 70,000, on SDS-gel electrophoresis. The zeta chain was eluted ahead of the other subunits during gel chromatography. It tended to dimerize during prolonged dialysis or purification procedures. The alpha and zeta chains were isolated in pure form. The gamma and delta chains, and also the beta and epsilon chains, were obtained as mixtures but were not separated from each other. All the subunits showed different antigenicities. The amino-terminal portions of the alpha through epsilon chains have the same sequence, Thr-Ile . Leu-Lys-Glu-Lys-Gln. The zeta chain has a different amino-terminal sequence, Val-Leu-Asp-X-Ile/Leu-Glu-Lys. The zeta chain contained only 1 mol of Cu/mol of protein, whereas the other chains contained 2 mol of Cu/mol of protein. No free sulfhydryl group was detected in th absence of guanidine. However, 3 mol of SH/mol of protein was detected immediately after the addition of 6 M guanidine-HCl. These SH groups were very unstable and disappeared on standing.