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Preparation of the polypeptides of the giant hemoglobin of Travisia japonica in carbon monoxy form

1982; Elsevier BV; Volume: 218; Issue: 2 Linguagem: Inglês

10.1016/0003-9861(82)90378-2

1096-0384

Kenzo Fushitani, H. Morimoto, Osamu Ochi,

Erythrocyte Function and Pathophysiology

Five heme-containing polypeptides of the giant hemoglobin of Travisia japonica (Polychaeta, Annelida) were separated in carbon monoxy form by ion-exchange chromatography. Their relationship to the four bands of native hemoglobin which are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-gel electrophoresis) with 2-mercaptoethanol was determined. The polypeptides were characterized by NH2-terminal amino acid analysis, SDS-gel electrophoresis and molecular weight determination. These polypeptides have been designated I, IIv, IIg, III, and IV. The terminal amino acids and SDS molecular weight were, respectively: Glu, 14,000; Val, 16,000; Gly, 16,000; Glu, 17,000; Glu, 18,000. The minimum molecular weight of these polypeptides, based on heme, ranged from 15,000 to 18,000. All the polypeptides showed reversible oxygen and carbon monoxide binding. Two-dimensional SDS-gel electrophoresis with and without 2-mercaptoethanol and NH2-terminal amino acid analysis of proteins in bands separated in SDS-gel electrophoresis without 2-mercaptoethanol showed that the lower molecular weight band 1 (Mr 12,000) without 2-mercaptoethanol gave polypeptides IIv (Val, 16,000) and III (Glu, 17,000). On the other hand, the higher molecular weight band 2 (54,000) without 2-mercaptoethanol gave polypeptides I (Glu, 14,000), IIg (Gly, 16,000), and IV (Glu, 18,000).