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. 1974 Jun;71(6):2222–2225. doi: 10.1073/pnas.71.6.2222

Noncovalent Modification of Deoxyhemoglobin S Solubility and Erythrocyte Sickling

Michael R Waterman 1, Kotaro Yamaoka 1,*, Larry Dahm 1, Joe Taylor 1, G L Cottam 1
PMCID: PMC388423  PMID: 4526343

Abstract

Ammonium sulfate, as well as potassium phosphate, can be used to measure solubility differences between hemoglobin S and hemoglobin A. In addation, the solubility of deoxyhemoglobin CHarlem in 1.96 M phosphate has a markedly different temperature dependence from that of deoxyhemoglobin S. This observation indicates that the solubility measurement is quite sensitive to changes in protein structure. Because of the large degree of comparability between the solubility and the aggregation of deoxyhemoglobin S, solubility was used to measure the effectiveness of organic compounds as noncovalent modifiers of deoxyhemoglobin S aggregation.

Organic solvents (ethanol, dimethylsulfoxide, 1,4-dioxane, dimethylformamide) alter the solubility characteristics of deoxyhemoglobin S in 1.96 M phosphate buffer, pH 7.0. The concentrations of solvent necessary to provide a half-maximal effect are remarkably similar (about 0.5 M), although the chemical nature of these compounds is quite different. The effect of these solvents must be to prevent the noncovalent bond formation necessary to produce the insoluble hemoglobin precipitate, perhaps by altering the water structure around the deoxyhemoglobin S molecules. In addition to these organic solvents, guanidine hydrochloride and urea, two well-known protein denaturants, were studied. Guanidine hydrochloride was as effective as the best organic solvent in increasing the solubility of deoxyhemoglobin S; urea was far less effective. Studies in vitro with intact erythrocytes from individuals homozygous for hemoglobin S showed that sickling is decreased up to 50% by treatment with ethanol. This offers further evidence that solubility is monitoring a phenomenon similar to the aggregation of deoxyhemoglobin S inside erythrocytes. While use of these particular compounds in vitro would seem to have no clinical implications, these studies do suggest that the use of chemicals that do not modify hemoglobin S covalently should be explored in efforts to prevent deoxyhemoglobin S aggregation.

Keywords: hemoglobin S, sickle cell disease, hemoglobin solubility

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Selected References

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

  1. Bookchin R. M., Nagel R. L., Ranney H. M. Structure and properties of hemoglobin C-Harlem, a human hemoglobin variant with amino acid substitutions in 2 residues of the beta-polypeptide chain. J Biol Chem. 1967 Jan 25;242(2):248–255. [PubMed] [Google Scholar]
  2. Bookchin R. M., Nagel R. L., Ranney H. M. The effect of beta 73 Asn on the interactions of sickling hemoglobins. Biochim Biophys Acta. 1970 Nov 17;221(2):373–375. doi: 10.1016/0005-2795(70)90279-5. [DOI] [PubMed] [Google Scholar]
  3. Cerami A., Manning J. M. Potassium cyanate as an inhibitor of the sickling of erythrocytes in vitro. Proc Natl Acad Sci U S A. 1971 Jun;68(6):1180–1183. doi: 10.1073/pnas.68.6.1180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cottam G. L., Waterman M. R. Reversible solubility of deoxyhemoglobin S. Biochem Biophys Res Commun. 1973 Oct 1;54(3):1157–1163. doi: 10.1016/0006-291x(73)90813-9. [DOI] [PubMed] [Google Scholar]
  5. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  6. Diederich D. Relationship between the oxygen affinity and in vitro sickling propensity of carbamylated sickle erythrocytes. Biochem Biophys Res Commun. 1972 Feb 16;46(3):1255–1261. doi: 10.1016/s0006-291x(72)80110-4. [DOI] [PubMed] [Google Scholar]
  7. Klotz I. M., Tam J. W. Acetylation of sickle cell hemoglobin by aspirin. Proc Natl Acad Sci U S A. 1973 May;70(5):1313–1315. doi: 10.1073/pnas.70.5.1313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kraus L. M., Kraus A. P. Carbamyl phosphate mediated inhibition of the sickling of erythrocytes in vitro. Biochem Biophys Res Commun. 1971 Sep 17;44(6):1381–1387. [PubMed] [Google Scholar]
  9. MURAYAMA M. Titratable sulfhydryl groups of normal and sickle cell hemoglobins at O degrees and 38 degrees. J Biol Chem. 1957 Sep;228(1):231–240. [PubMed] [Google Scholar]
  10. May A., Bellingham A. J., Huehns E. R., Beaven G. H. Effect of cyanate on sickling. Lancet. 1972 Mar 25;1(7752):658–661. doi: 10.1016/s0140-6736(72)90462-x. [DOI] [PubMed] [Google Scholar]
  11. ORNSTEIN L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964 Dec 28;121:321–349. doi: 10.1111/j.1749-6632.1964.tb14207.x. [DOI] [PubMed] [Google Scholar]
  12. Roth E. F., Jr, Nagel R. L., Bookchin R. M., Grayzel A. I. Nitrogen mustard: an "in vitro" inhibitor of erythrocyte sickling. Biochem Biophys Res Commun. 1972 Aug 7;48(3):612–618. doi: 10.1016/0006-291x(72)90392-0. [DOI] [PubMed] [Google Scholar]

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