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. 1983 Sep;72(3):846–852. doi: 10.1172/JCI111055

Intracellular polymerization of sickle hemoglobin. Effects of cell heterogeneity.

C T Noguchi, D A Torchia, A N Schechter
PMCID: PMC1129249  PMID: 6886006

Abstract

To determine the extent to which the broad distribution in intracellular hemoglobin concentrations found in sickle erythrocytes affects the extent of intracellular polymerization of hemoglobin S, we have fractionated these cells by density using discontinuous Stractan gradients. The amount of polymer formed in the subpopulations was experimentally measured as a function of oxygen saturation using 13C nuclear magnetic resonance spectroscopy. The results for each subpopulation are in very good agreement with the theoretical predictions based on the current thermodynamic description for hemoglobin S gelation. We further demonstrate that the erythrocyte density profile for a single individual with sickle cell anemia can be used with the theory to predict the amount of polymer in unfractionated cells. We find that heterogeneity in intracellular hemoglobin concentration causes the critical oxygen saturation for formation of polymer to shift from 84 to greater than 90%; polymer is formed predominantly in the dense cells at the very high oxygen saturation values. The existence of polymer at arterial oxygen saturation values has significance for understanding the pathophysiology of sickle cell anemia. The utility of these techniques for assessing various therapeutic strategies is discussed.

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Selected References

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  1. Abraham E. C., Walker D., Gravely M., Huisman T. H. Minor hemoglobins in sickle cell anemia, beta-thalassemia, and related conditions: a study of red cell fractions isolated by density gradient centrifugation. Biochem Med. 1975 May;13(1):56–77. doi: 10.1016/0006-2944(75)90140-4. [DOI] [PubMed] [Google Scholar]
  2. Bunn H. F., Noguchi C. T., Hofrichter J., Schechter G. P., Schechter A. N., Eaton W. A. Molecular and cellular pathogenesis of hemoglobin SC disease. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7527–7531. doi: 10.1073/pnas.79.23.7527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clark M. R., Greenquist A. C., Shohet S. B. Stabilization of the shape of sickled cells by calcium and A23187. Blood. 1976 Dec;48(6):899–909. [PubMed] [Google Scholar]
  4. Clark M. R., Mohandas N., Embury S. H., Lubin B. H. A simple laboratory alternative to irreversibly sickled cell (ISC) counts. Blood. 1982 Sep;60(3):659–662. [PubMed] [Google Scholar]
  5. Clark M. R., Mohandas N., Shohet S. B. Deformability of oxygenated irreversibly sickled cells. J Clin Invest. 1980 Jan;65(1):189–196. doi: 10.1172/JCI109650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Coletta M., Hofrichter J., Ferrone F. A., Eaton W. A. Kinetics of sickle haemoglobin polymerization in single red cells. Nature. 1982 Nov 11;300(5888):194–197. doi: 10.1038/300194a0. [DOI] [PubMed] [Google Scholar]
  7. Corash L. M., Piomelli S., Chen H. C., Seaman C., Gross E. Separation of erythrocytes according to age on a simplified density gradient. J Lab Clin Med. 1974 Jul;84(1):147–151. [PubMed] [Google Scholar]
  8. Dover G. J., Boyer S. H., Pembrey M. E. F-cell production in sickle cell anemia: regulation by genes linked to beta-hemoglobin locus. Science. 1981 Mar 27;211(4489):1441–1444. doi: 10.1126/science.6162200. [DOI] [PubMed] [Google Scholar]
  9. Fabry M. E., Kaul D. K., Raventos-Suarez C., Chang H., Nagel R. L. SC erythrocytes have an abnormally high intracellular hemoglobin concentration. Pathophysiological consequences. J Clin Invest. 1982 Dec;70(6):1315–1319. doi: 10.1172/JCI110732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fabry M. E., Nagel R. L. The effect of deoxygenation on red cell density: significance for the pathophysiology of sickle cell anemia. Blood. 1982 Dec;60(6):1370–1377. [PubMed] [Google Scholar]
  11. Ferrone F. A., Hofrichter J., Sunshine H. R., Eaton W. A. Kinetic studies on photolysis-induced gelation of sickle cell hemoglobin suggest a new mechanism. Biophys J. 1980 Oct;32(1):361–380. doi: 10.1016/S0006-3495(80)84962-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gill S. J., Spokane R., Benedict R. C., Fall L., Wymann J. Ligand-linked phase equilibria of sickle cell hemoglobin. J Mol Biol. 1980 Jun 25;140(2):299–312. doi: 10.1016/0022-2836(80)90107-2. [DOI] [PubMed] [Google Scholar]
  13. Goldberg M. A., Lalos A. T., Bunn H. F. The effect of erythrocyte membrane preparations on the polymerization of sickle hemoglobin. J Biol Chem. 1981 Jan 10;256(1):193–197. [PubMed] [Google Scholar]
  14. Hofrichter J. Ligand binding and the gelation of sickle cell hemoglobin. J Mol Biol. 1979 Mar 5;128(3):335–369. doi: 10.1016/0022-2836(79)90092-5. [DOI] [PubMed] [Google Scholar]
  15. Kowalczykowski S., Steinhardt J. Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity. J Mol Biol. 1977 Sep 15;115(2):201–213. doi: 10.1016/0022-2836(77)90097-3. [DOI] [PubMed] [Google Scholar]
  16. Minton A. P. Non-ideality and the thermodynamics of sickle-cell hemoglobin gelation. J Mol Biol. 1977 Feb 15;110(1):89–103. doi: 10.1016/s0022-2836(77)80100-9. [DOI] [PubMed] [Google Scholar]
  17. Noguchi C. T., Schechter A. N. The intracellular polymerization of sickle hemoglobin and its relevance to sickle cell disease. Blood. 1981 Dec;58(6):1057–1068. [PubMed] [Google Scholar]
  18. Noguchi C. T., Torchia D. A., Schechter A. N. Determination of deoxyhemoglobin S polymer in sickle erythrocytes upon deoxygenation. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5487–5491. doi: 10.1073/pnas.77.9.5487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Noguchi C. T., Torchia D. A., Schechter A. N. Polymerization of hemoglobin in sickle trait erythrocytes and lysates. J Biol Chem. 1981 May 10;256(9):4168–4171. [PubMed] [Google Scholar]
  20. Oda S., Oda E., Tanaka K. R. Relationship of density distribution and pyruvate kinase electrophoretic pattern of erythrocytes in sickle cell diseases and other disorders. Acta Haematol. 1978;60(4):201–209. doi: 10.1159/000207716. [DOI] [PubMed] [Google Scholar]
  21. Ross P. D., Briehl R. W., Minton A. P. Temperature dependence of nonideality in concentrated solutions of hemoglobin. Biopolymers. 1978 Sep;17(9):2285–2288. doi: 10.1002/bip.1978.360170920. [DOI] [PubMed] [Google Scholar]
  22. Rossi-Bernardi L., Perella M., Luzzana M., Samaja M., Raffaele I. Simultaneous determination of hemoglobin derivatives, oxygen content, oxygen capacity, and oxygen saturation in 10 microliters of whole blood. Clin Chem. 1977 Jul;23(7):1215–1225. [PubMed] [Google Scholar]
  23. Sunshine H. R., Hofrichter J., Eaton W. A. Gelation of sickle cell hemoglobin in mixtures with normal adult and fetal hemoglobins. J Mol Biol. 1979 Oct 9;133(4):435–467. doi: 10.1016/0022-2836(79)90402-9. [DOI] [PubMed] [Google Scholar]
  24. Sunshine H. R., Hofrichter J., Ferrone F. A., Eaton W. A. Oxygen binding by sickle cell hemoglobin polymers. J Mol Biol. 1982 Jun 25;158(2):251–273. doi: 10.1016/0022-2836(82)90432-6. [DOI] [PubMed] [Google Scholar]
  25. Sutherland J. W., Egan W., Schechter A. N., Torchia D. A. Carbon-13-proton nuclear magnetic double-resonance study of deoxyhemoglobin S gelation. Biochemistry. 1979 May 1;18(9):1797–1803. doi: 10.1021/bi00576a025. [DOI] [PubMed] [Google Scholar]
  26. Vettore L., De Matteis M. C., Zampini P. A new density gradient system for the separation of human red blood cells. Am J Hematol. 1980;8(3):291–297. doi: 10.1002/ajh.2830080307. [DOI] [PubMed] [Google Scholar]

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