Hemocyanins in Spiders, VI. Comparison of the Polypeptide Chainsof Eurypelma californicumHemocyanin

Abstract

The subunits of the hemocyanin from the tarantula, E. californicum, were isolated following dissociation at pH 9.6, by a sequence of chromatographic and electrophoretic steps. Fraction 2 (containing 2 chains, a and c2) and the constituent polypeptide chains of the dimeric subunit 4D (b and c4) were resolved by anion exchange chromatography at pH 8.9 and 6.5, respectively. Since c2 and c4 have different electrophoretic mobilities in polyacrylamide gradient gels, the total number of different polypeptide chains is 7. The amino acid compositions of the 7 chains are reported. There are major differences for at least half of the amino acids, while more consistent proportions become evident if the amino acids are grouped by types of side chains. The N-terminal amino acid is proline in chains b and e, while no end group could be detected in any of the other chains by different methods. The C-terminal end group was valine in chains d and e. Cleavage by 70% formic acid, and by cyanogen bromide in formic acid, results in fragmentation patterns distinct for each chain. After cyanogen bromide cleavage, the 2 largest peptides of each chain are of MW near 24,000. Tryptic fingerprints also reveal significant differences between all chains. Subunit heterogeneity of Eurypelma hemocyanin is clearly not the consequence of secondary modifications but resides in major differences of the amino acid sequences.