The dissociation of carbon monoxide from hemoglobin intermediates

M. Samaja, E. Rovida, M. Niggeler, M. Perrella, L. Rossi-Bernardi

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Abstract

To investigate the mechanism of allosteric switching in human hemoglobin, we have studied the dissociation of the ligand (CO) from several intermediate ligation states by a stopped-flow kinetic technique that utilizes competitive binding of CO by microperoxidase. The hemoglobin species investigated include Hb(CO)4, the diliganded symmetrical species (α β-CO)2 and (α-CO β)2 and the di- and monoliganded asymmetrical species (α-CO β-CO)(α β), (α-CO β)(α β-CO), (α β-CO)(α β), and (α-CO β)(α β). They were obtained by rapid reduction with dithionite of the corresponding valence intermediates that in turn were obtained by chromatography or by hybridization. The nature and concentration of the intermediates were determined by isoelectric focusing at -25°C. The study was performed at varying hemoglobin concentrations (0.1, 0.02, and 0.001 mM [heme]), pH (6.0, 7.0, 8.0), with and without inositol hexaphosphate. The results indicate that: (a) hemoglobin concentration in the 0.1-0.02 mM range does not significantly affect the kinetic rates; (b) the α chains dissociate CO faster than the β chains; (c) the symmetrical diliganded intermediates show cooperativity with respect to ligand dissociation that disappears in the presence of inositol hexaphosphate; (d) the monoliganded intermediates dissociate CO faster than the diliganded intermediates; (e) the asymmetrical diliganded intermediates are functionally different from the symmetrical species.

Original languageEnglish
Pages (from-to)4528-4533
Number of pages6
JournalJournal of Biological Chemistry
Volume262
Issue number10
Publication statusPublished - 1987

ASJC Scopus subject areas

  • Biochemistry

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    Samaja, M., Rovida, E., Niggeler, M., Perrella, M., & Rossi-Bernardi, L. (1987). The dissociation of carbon monoxide from hemoglobin intermediates. Journal of Biological Chemistry, 262(10), 4528-4533.