Nitros-ylhemoglobin (Hb-NO), prepared in a gas-tight syringe anaerobically, was reacted with dissolved oxygen (O
2) in phosphate buffered saline at pH 7.4 and 37°C. The reaction process was monitored by measuring (i) O
2 concentration in the medium by O
2 electrode, continuously, (ii) Hb-NO and methemoglobin (Met Hb) concentrations by EPR spectroscopy and (iii) NO
2- and NO
3- concentrations by chemical analyses.
(1) Hb-NO was quantitatively converted to Met Hb by consuming dissolved O
2, and NO
3- was produced.The reaction process consisted of branched, successive reactions:(i) The dissociation of Hb-NO was rate-limiting step, yielding deoxyhemoglobin (deoxy Hb) and NO; NO in solution changed to NO
2-.(ii) At higher O
2concentration, oxyhemoglobin (Hb-O
2) was transiently formed, and reacted with NO
2-, yielding Met Hb and NO
3-.(iii) At lower O
2 concentration, deoxy Hb reacted with NO
2-. yielding Hb-NO, Met Hb and NO
3-.(iv) NO
3- production from NO
2- and O
2 was catalyzed by Met Hb. The following scheme was proposed.
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(2) As proceeding the Met Hb formation from Hb-NO, the ratio of α-NO to β-NO in Hb-NO augumented and the alteration of α-NO was found, as judged from ESR spectra.
(3) The reaction between Hb-NO in erythrocytes and dissolved O
2 was qualitatively interpreted by the proposed scheme.
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