カンケイ ドウブツ アオゴカイ Perinereis aibuhitensis キョダイ ヘモグロビン ノ グロビンサ ニ フクマレル ケウナ SSケツゴウ

The extracellular hemoglobin (Hb) from the polychaete Perinereis aibuhitensis consists of four types of 144 globins and two types of 36 linker chains，having a molecular mass of about 3,500 kDa. There are two types of globin subunits: monomer chain a and disulfide-bondedt rimer AbB. The amino acid sequences of the four globin chains (a，A ，b ，B ) were already reported，previously (Yamanaka，M. et al. Natural Science Research of Tokushima University, 19, 63-92, 2005). The site of disulfide bonds in the globin subunits have been investigated. Each globin chain contains an intrachain disulfide bond between N-terminal and C-terminal Cys residues. In addition，the interchain disulfide bonds were found between chains A and b，and b and B. Therefore，it is elucidated that the chain b is situated at the center of disulfide-bonded trimer，such as A-b-B.The sites of disulfide-bonds determined all could be suitably fitted to the tree dimensional structure of each subunit in a model without stretching or twisting. It was also confirmed that there is no free Cys residue in Peinereis globins. The positions of Cys residues of Perinereis globin sequences were compared wIth those of other 27 chains derived from the homologous Hbs. Among 31 sequences，Cys residues were distributed in six sites. The sites 1 and 2 are located at the N-terminal region of amino acid sequences，the sItes 3 and 4 at the central region，and the sites 5 and 6 at C-terminal region. Furthermore，the Cys distribution was categorized into eight patterns. Perinereis Hb has four patterns I，IV，VII，VIII，be ing lack of the central sites 3 and 4. It should be noted that the pattern II includes the unique globins from Hbs of Lamellibrachia，Riftia and Oligobrachia that carry H2S to the symbiotic bacteria，suggesting that these globin chains might carry H2S in vivo. The phylogenetic tree of 31 globin chains derived from the giant Hbs is divided into two families A and B， as already poited out by us Previously. The family A indudes pattern I-V，whereas the family B includes V- VIII

Reassociation of annelid giant hemoglobin from the polychaete Perinereis aibuhitensis

Annelid extracellular hemoglobin (Hb) is a supramolecule with molecular mass of ～3,500kDa. The giant Hb consists of 12 subassemblies (globin dodecamers, D) and 18 homodimeric linkers (L) of non-globin chain. The globin dodecamer and linker were isolated from the polychaete Perinereis aibuhittensis Hb separately. Subsequently, these two components were mixed in the presence of 1M urea at a neutral pH to reform a whole molecule of Hb. At first L was refined by reverse phase chromatography in organic solvent. On the other hand, Perinereis Hb was incubated in 4M urea solution at 4°C for 5 min, and applied to two amphoteric ion-exchange resin column to remove L stick to the resin, and to isolate only D. The eluate was condensed and subjected to gel filtration. As a result, an ingredient of molecule mass ～210 kDa, that is D, was provided in high yield. When D and L were mixed in the molar ratio of approximately 1:1 in 50mM phosphate buffer (pH 7.2) in the presence of 1 M urea at room temperature, most of the proteins met to natural Hb size again within about 20 hours. Furthermore, similar experiments were performed in 50 mM Tris-HCl buffer (pH 7.2) containing 1 M urea in the presence of 1 mM CaCl2 or 1mM EDTA. It was observed that the reassociation was affected substantially by the presence of Ca2+. In conclusion, the homodimeric linkers have the key role to form the gigantic Hb