| Research Abstract |
An annelid extracellular hemoglobin (Hb) has a molecular mass of 3.5 MDa, which is ca. 50 times larger than human Hb and doubles in size of a ribosome. It is known that there are four species of globin chains (a, A, b, B) and two types of non-heme proteins (linkers ; L1, L2) in the giant Hb. The globin subunits form 12 substructures which are linked by the linker chains. In the present study, we focused on the subunit assembly, role of linker, and binding manner between globin substructures and linkers. Main results obtained are as follows :
1. Molecular masses of substructures are determined directly for some annelid Hbs by ESI-MS. The mass values obtained have revealed that the substructures are composed of globin dodecamer (3 (a, A, b, B)).
2. It was found that linker chains are preferentially digested by some proteolytic enzymes, remaining globin substructures to be intact. Thus, the substructures were isolated and their molecular masses were estimated by light scattering.
3. Amino acid sequences of chains a, b, and B of Perinereis Hb were determined. The molecular masses obtained from the sequences are in good agreement with the values of ESI-MS. This leads us to conclude that these globin chains do not contain any carbohydrate. Phylogenetic tree showed that there are two globin strains in annelid Hbs which were diversed about the same era when vertebrate myoglobin and hemoglobin were diversed.
4. It was demonstrated that linkers has a structuring activity to form a giant Hb in the final stage of subunit assembly.
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