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. 1981 Aug;35(2):265–270. doi: 10.1016/S0006-3495(81)84788-1

Tetramer-dimer dissociation of carboxyhemoglobin in the absence of dithionite.

C A Sawicki, Q H Gibson
PMCID: PMC1327521  PMID: 7272439

Abstract

The generally accepted value for the tetramer-dimer dissociation constant KL4,2 of carboxyhemoglobin in pH 7.0 phosphate buffer lies in the range 1--2 micrometers. Previous determinations of the quantity have generally involved addition of dithionite to samples to exclude oxygen. We report flash photolysis experiments on carboxyhemoglobin in the absence of dithionite which suggest that KL4,2 is 0.2 +/- 0.05 micrometer. Addition of dithionite to our samples resulted in an order of magnitude increase in KL4,2 in good agreement with previously published results. The mechanism of this increase in dissociation has not been determined with certainty. However, impurities, possibly metal ions, are required in addition to dithionite to produce this effect. Dithionite did not increase KL4,2 for phosphate buffer solutions treated with Chelex 100 analytical grade chelating resin. Addition of bovine serum albumin to untreated buffer solutions before addition of dithionite was found to prevent increased dissociation. The sulfhydryl-reducing agents dithiothreitol and beta-mercaptoethanol were found to protect against the effect of dithionite and to reverse its effect on KL4,2 if they were added after the dithionite. The interaction of the unknown impurities with dithionite to produce increased values of KL4,2 could be mimicked by addition of CU2+ ions in concentrations of less than 1 micrometer to buffer treated with Chelex 100 resin.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersen M. E., Moffat J. K., Gibson Q. H. The kinetics of ligand binding and of the association-dissociation reactions of human hemoglobin. Properties of deoxyhemoglobin dimers. J Biol Chem. 1971 May 10;246(9):2796–2807. [PubMed] [Google Scholar]
  2. DeSa R. J., Gibson Q. H. A practical automatic data acquisition system for stopped-flow spectrophotometry. Comput Biomed Res. 1969 Oct;2(5):494–505. doi: 10.1016/0010-4809(69)90014-7. [DOI] [PubMed] [Google Scholar]
  3. Edelstein S. J., Rehmar M. J., Olson J. S., Gibson Q. H. Functional aspects of the subunit association-dissociation equilibria of hemoglobin. J Biol Chem. 1970 Sep 10;245(17):4372–4381. [PubMed] [Google Scholar]
  4. GIBSON Q. H. The direct determination of the velocity constant of the reaction Hb4 (CO)3 + CO-Hb4(CO)4. J Physiol. 1956 Oct 29;134(1):123–134. doi: 10.1113/jphysiol.1956.sp005628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gray R. D. The effect of 2,3-diphosphoglycerate on the tetramer-dimer equilibrium of liganded hemoglobin. J Biol Chem. 1974 May 10;249(9):2879–2885. [PubMed] [Google Scholar]
  6. Gray R. D. The effect of H+, inositol hexaphosphate, and Zn(II) on the tetramer-dimer equilibrium of liganded hemoglobin. J Biol Chem. 1980 Mar 10;255(5):1812–1818. [PubMed] [Google Scholar]
  7. Guidotti G. Studies on the chemistry of hemoglobin. II. The effect of salts on the dissociation of hemoglobin into subunits. J Biol Chem. 1967 Aug 25;242(16):3685–3693. [PubMed] [Google Scholar]
  8. Kellett G. L., Gutfreund H. Reactions of haemoglobin dimers after ligand dissociation. Nature. 1970 Aug 29;227(5261):921–926. doi: 10.1038/227921a0. [DOI] [PubMed] [Google Scholar]
  9. Saffran W. A., Gibson Q. H. Photodissociation of ligands from heme and heme proteins. Effect of temperature and organic phosphate. J Biol Chem. 1977 Nov 25;252(22):7955–7958. [PubMed] [Google Scholar]
  10. Sawicki C. A., Gibson Q. H. Properties of the T state of human oxyhemoglobin studies by laser photolysis. J Biol Chem. 1977 Nov 10;252(21):7538–7547. [PubMed] [Google Scholar]
  11. Sawicki C. A., Gibson Q. H. Quaternary conformational changes in human oxyhemoglobin studied by laser photolysis. J Biol Chem. 1977 Aug 25;252(16):5783–5788. [PubMed] [Google Scholar]
  12. Wiedermann B. L., Olson J. S. Acceleration of tetramer formation by the binding of inositol hexaphosphate to hemoglobin dimers. J Biol Chem. 1975 Jul 10;250(13):5273–5275. [PubMed] [Google Scholar]
  13. Willard J. M., Davis J. J., Wood H. G. Phosphoenolpyruvate carboxytransphosphorylase. IV. Requirement for metal cations. Biochemistry. 1969 Aug;8(8):3137–3144. doi: 10.1021/bi00836a002. [DOI] [PubMed] [Google Scholar]

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