Subunit structure of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus and amino acid sequence of the constituent polypeptide chain (IIC).

Tylorrhynchus cyanomethemoglobin reduced with dithiothreitol was separated by chromatofocusing into four heme-containing polypeptide chains (I, IIA, IIB, and IIC) and a non-heme chain (N). The molecular weights of chains IIA-C and N were confirmed to be the same by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on a 10-20% gradient gel. The molecular weight of chain IIC was determined to be 17,415 (including heme) from the amino acid sequence. Chain N constitutes less than 5% of the total protein and has the same NH2-terminal sequence, suggesting that it is derived from chain IIA during the isolation procedure. Tylorrhynchus hemoglobin consists of two types of subunit with molecular weights of 16,327 (chain I) and approximately 50,000, and the latter splits into chains IIA-C in the presence of a reducing agent. On the basis of the accurate value obtained for the molecular mass of chain IIC, it was concluded that the subunit of approximately 50,000 daltons is a trimer of heme-containing chains IIA, IIB, and IIC linked by disulfide bonds. The cysteine residue at position 5 and the arginine at position 10 are conserved in the four heme-containing chains of Tylorrhynchus hemoglobin. The complete sequence of 149 residues of Tylorrhynchus chain IIC was determined. This sequence shows high homology with Tylorrhynchus chain I (Suzuki, T., Takagi, T., and Gotoh, T. (1982) Biochem. Biophys. Acta 708, 253-258) and Lumbricus chain AIII (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015).