The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality.
The dissociation of the extracellular hemoglobin of Tubifex tubifex at alkaline and acid pH, and its reassociation upon return to neutral pH, was investigated using gel filtration, ultracentrifugation, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Tubifex hemoglobin dissociated at pH above 8 and below 6; both dissociations appeared to be equilibrium processes. The extent of dissociation increased as the pH moved away from neutrality; although dissociation was virtually complete at pH 11, its extent at acid pH did not exceed 50-60% at pH 4. Ca(II), Mg(II), and Sr(II) cations over the range 1-100 mM decreased the extent of the dissociation only at alkaline pH. The visible absorption spectrum of the oxyhemoglobin remained unaltered in the pH range 4-9. At more extreme pH, it changed with time, altering irreversibly to that of the aquo ferri form. Gel filtration of the hemoglobin at both extremes of pH showed that it dissociated into two heme-containing fragments; one consisting of subunit 1 (Mr approximately 17,000) and the other containing subunits 2, 3, and 4 of the hemoglobin (Mr approximately 60,000). Upon return to neutral pH, the dissociated fragment reassociated to the extent of 50 to 80% to whole hemoglobin molecules. The reassociation decreased with increase in alkaline pH, and with decrease in acid pH to which the hemoglobin had been exposed; it increased in the presence of Ca(II), Sr(II), and Mg(II) only subsequent to dissociation at alkaline pH. The SDS-PAGE patterns, gel-filtration elution volumes, and alpha-helical contents, determined from circular dichroism at 222 nm, of the reassociated whole molecules were identical to those of the native hemoglobin.
D000818:Animals; D002413:Cations, Divalent; D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006454:Hemoglobins; D006863:Hydrogen-Ion Concentration; D009835:Oligochaeta / Q000032:analysis*; D010108:Oxyhemoglobins; D010446:Peptide Fragments; D011487:Protein Conformation; D013053:Spectrophotometry; D013997:Time Factors
D002413:Cations, Divalent; D006454:Hemoglobins; D010108:Oxyhemoglobins; D010446:Peptide Fragments