Abstract
We report the results of a kinetic investigation on the gelation of purified deoxyhemoglobin S. Gelation was induced by raising the temperature and was monitored by measuring both the heat absorbed, with a microcalorimeter, and the appearance of linear birefringence, with a microspectrophotometer. The kinetics are unusual. Prior to the onset of gelation there is a delay period, followed by a sigmoidal progress curve. The delay time is formally dependent on approximately the 30th power of the deoxyhemoglobin S concentration; a decrease in concentration from 23 to 22 g/dl increases the delay time by a factor of four. It is also extremely temperature dependent; a 1°C temperature rise in the range 20-30°C almost halves the delay time. From these results we conclude that the initial rate is controlled by the nucleation of individual fibers. We present a kinetic model that accounts for the concentration, temperature, and time dependence of the initial phase of the gelation reaction. Extrapolation of our data to physiological conditions predicts that changes in intracellular hemoglobin concentration and oxygen saturation, realizable in vivo, produce enormous changes in the delay time. The range of delay times spans both the mean capillary transit and total circulation times. This result points to the delay time as an extremely important variable in determining the course of sickle cell disease, and suggests a new approach to therapy.
Keywords: calorimetry, pathophysiology, nucleation theory, sickle cell therapy, macromolecular assembly
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Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- ALLISON A. C. Observations on the sickling phenomenon and on the distribution of different haemoglobin types in erythrocyte populations. Clin Sci. 1956 Nov;15(4):497–510. [PubMed] [Google Scholar]
- ALLISON A. C. Properties of sickle-cell haemoglobin. Biochem J. 1957 Feb;65(2):212–219. doi: 10.1042/bj0650212. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bertles J. F., Rabinowitz R., Döbler J. Hemoglobin interaction: modification of solid phase composition in the sickling phenomenon. Science. 1970 Jul 24;169(3943):375–377. doi: 10.1126/science.169.3943.375. [DOI] [PubMed] [Google Scholar]
- Bookchin R. M., Nagel R. L. Conformational requirements for the polymerization of hemoglobin S: studies of mixed liganded hybrids. J Mol Biol. 1973 May 15;76(2):233–239. doi: 10.1016/0022-2836(73)90387-2. [DOI] [PubMed] [Google Scholar]
- Bookchin R. M., Nagel R. L. Ligand-induced conformational dependence of hemoglobin in sickling interactios. J Mol Biol. 1971 Sep 14;60(2):263–270. doi: 10.1016/0022-2836(71)90292-0. [DOI] [PubMed] [Google Scholar]
- Briehl R. W., Ewert S. Effects of pH, 2,3-diphosphoglycerate and salts on gelation of sickle cell deoxyhemoglobin. J Mol Biol. 1973 Nov 5;80(3):445–458. doi: 10.1016/0022-2836(73)90415-4. [DOI] [PubMed] [Google Scholar]
- CHARACHE S., CONLEY C. L. RATE OF SICKLING OF RED CELLS DURING DEOXYGENATION OF BLOOD FROM PERSONS WITH VARIOUS SICKLING DISORDERS. Blood. 1964 Jul;24:25–48. [PubMed] [Google Scholar]
- Eaton W. A., Lewis T. P. Polarized single-crystal absorption spectrum of 1-methyluracil. J Chem Phys. 1970 Sep 15;53(6):2164–2172. doi: 10.1063/1.1674310. [DOI] [PubMed] [Google Scholar]
- Finch J. T., Perutz M. F., Bertles J. F., Döbler J. Structure of sickled erythrocytes and of sickle-cell hemoglobin fibers. Proc Natl Acad Sci U S A. 1973 Mar;70(3):718–722. doi: 10.1073/pnas.70.3.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hofrichter J., Hendricker D. G., Eaton W. A. Structure of hemoglobin S fibers: optical determination of the molecular orientation in sickled erythrocytes. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3604–3608. doi: 10.1073/pnas.70.12.3604. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huisman T. H., Dozy A. M. Studies on the heterogeneity of hemoglobin. IX. The use of Tris(hydroxymethyl)aminomethanehcl buffers in the anion-exchange chromatography of hemoglobins. J Chromatogr. 1965 Jul;19(1):160–169. doi: 10.1016/s0021-9673(01)99434-8. [DOI] [PubMed] [Google Scholar]
- Konotey-Ahulu F. I. The sickle cell diseases. Clinical manifestations including the "sickle crisis". Arch Intern Med. 1974 Apr;133(4):611–619. [PubMed] [Google Scholar]
- Magdoff-Fairchild B., Swerdlow P. H., Bertles J. F. Intermolecular organization of deoxygenated sickle haemoglobin determined by x-ray diffraction. Nature. 1972 Sep 22;239(5369):217–219. doi: 10.1038/239217a0. [DOI] [PubMed] [Google Scholar]
- Malfa R., Steinhardt J. A temperature-dependent latent-period in the aggregation of sickle-cell deoxyhemoglobin. Biochem Biophys Res Commun. 1974 Aug 5;59(3):887–893. doi: 10.1016/s0006-291x(74)80062-8. [DOI] [PubMed] [Google Scholar]
- Messer M. J., Harris J. W. Filtration characteristics of sickle cells: rates of alteration of filterability after deoxygenation and reoxygenation, and correlations with sickling and unsickling. J Lab Clin Med. 1970 Oct;76(4):537–547. [PubMed] [Google Scholar]
- Minton A. P. A thermodynamic model for gelation of sickle-cell hemoglobin. J Mol Biol. 1974 Feb 5;82(4):483–498. doi: 10.1016/0022-2836(74)90243-5. [DOI] [PubMed] [Google Scholar]
- SAUNDERS M., ROSS P. D. A simple model of the reaction between polyadenylic acid and polyuridylic acid. Biochem Biophys Res Commun. 1960 Sep;3:314–318. doi: 10.1016/0006-291x(60)90248-5. [DOI] [PubMed] [Google Scholar]
- Seakins M., Gibbs W. N., Milner P. F., Bertles J. F. Erythrocyte Hb-S concentration. An important factor in the low oxygen affinity of blood in sickle cell anemia. J Clin Invest. 1973 Feb;52(2):422–432. doi: 10.1172/JCI107199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Williams R. C., Jr Concerted formation of the gel of hemoglobin S. Proc Natl Acad Sci U S A. 1973 May;70(5):1506–1508. doi: 10.1073/pnas.70.5.1506. [DOI] [PMC free article] [PubMed] [Google Scholar]