アオゴカイ巨大ヘモグロビンの再構成

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  • アオゴカイ キョダイ ヘモグロビン ノ サイコウセイ
  • Reassociation of annelid giant hemoglobin from the polychaete Perinereis aibuhitensis

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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.

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