Purification and structure of the polypeptide chains of earthworm hemoglobin

Abstract Carboxyhemoglobin from the earthworm, Lumbricus terrestris , separates on isoelectric focusing into a major component A, and a minor component B, which comprises 4–9% of the total. The molecular weights of all the polypeptide chains from either component have been estimated to be near 15,000–16,000 by chromatography on Sephacryl S-200 in 6 m guanidine-HCl after oxidation with performic acid. Species of higher molecular weight were not detected under these conditions. The chains remain partially aggregated, however, in 8 m urea. Electrophoresis in 8 m urea at pH 3.5 on disc gels results in the separation of four protein bands. Analysis of chromatography of either globin A or B on carboxymethylcellulose in 8 m urea indicates that three of these bands are unique polypeptides chains. The fourth, most anodic, band appears to be a product of aggregation and not a unique polypeptide chain. The amino acid composition has been determined for the three chains isolated from each component. The NH 2 -terminal residues for the three isolated chains of globin A have been determined to be aspartic acid, alanine, and lysine. The unfractionated globin and that from components A and B have the same NH 2 termini.