The giant, extracellular heme-containing respiratory proteins of annelids possess a characteristic hexagonal bilayer appearance in election micrographs, have a molecular weight of ca. 4x106, and contain about 160 heme groups. The hemoglobin of the earthworm Lumbricus terrestris is the most studied of this group: it consists of four subunits M (chain I, 16.7 kDa), Da (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (disulfide bonded chains II, III, and IV, 53 kDa). Chains I through IV contain one heme per ca. 17 kDa and chain VI contains one heme per 32 kDa. The chlorocruorin from the marine polychaete Myxicola infundibulum contain an altered heme group with a formyl instead of a vinyl side-chain configuration. The chlorocruorins consist of two types of subunits D (one or more chains of 35 kDa) and T* (a disulfide bonded tetramer of chains I-IV, each ca. 17 kDa). We have completed the amino acid sequences of chain I from Lumbricus and the corresponding chain of another oligochaete Tubifex tubifex. We have also determined that the N-terminal sequences of Lumbricus chains I through VI and of the corresponding chains of hemoglobin from the marine worm Tylorrhynchus heterochaetus strongly suggests that there are two strains of these globin chains, comparable to the relationship of the alpha and beta chains of vertebrate hemoglobins. A primary aim of the proposed research will be to obtain the amino acid sequences of Lumbricus hemoglobin chains V and VI (D1 and D2) as well as the T* chains of the chlorocruorin from Myxicola. In addition, we will complete the sequence of the trimer chains of the Tubifex hemoglobin and the heme-containing chains of the leech, Macrobdella. These will all be done by direct chemical analysis. In addition, we will establish the cDNA lambda gt10 libraries for the globin genes of the chlorocruorin of the polychaete Myxicola and the hemoglobin of Lumbricus. Our broad aim is to relate the primary structure of the annelid hemoglobins and chlorocruorins to one another as well as to the recently proposed model, wherein the D subunits either form a circular scaffolding decorated with complexes of T and M (or T*) subunits or act as linkers between the latter.
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