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pubmed:abstractText |
We have measured the forward and reverse rates of the allosteric transition between R (relaxed) and T (tense) quaternary structures for oxyhemoglobin A from which a single oxygen molecule was removed in pH 7, phosphate buffer, using the method of modulated excitation (Ferrone, F.A., and J.J. Hopfield. 1976. Proc. Natl. Acad. Sci. USA. 73:4497-4501 and Ferrone, F.A., A.J. Martino, and S. Basak. 1985. Biophys. J. 48:269-282). Despite the low quantum yield, which necessitated large light levels and an associated temperature rise, the data was of superior quality to the equivalent experiment with CO as a ligand, permitting comparison between the allosteric behavior of hemoglobin with different ligands. Qualitatively, the T structure is favored more strongly in triligated oxyhemoglobin than triligated carboxyhemoglobin. The rates for the allosteric transition with oxygen bound were essentially temperature independent, whereas for CO both the R----T and T----R rates increased with temperature, having an activation energy of 2.2 and 2.8 kcal, respectively. The R----T rate was higher for O2 than for CO being 3 x 10(3) s-1 vs. 1.6 x 10(3) s-1 for HbCO at 25 degrees C. The T----R rate for HbO2 was only 2 x 10(3) s-1, vs 4.2 x 10(3) s-1 for HbCO, giving an equilibrium constant between the structures greater than unity (L3 = 1.5). The data suggest that there may be some allosteric inequality between the subunits, but do not require (or rule out) ligand binding heterogeneity. The ligand-dependent differences are compatible with stereochemical studies of HbCO and HbO2. However,the large population of T species with three oxygen molecules bound is much greater than predicted by precision equilibrium studies and a generalized Szabo-Karplus model (Lee, A. W., M. Karplus, C. Poyart, and E. Bursaux. 1988. Biochemistry.27:1285-1301) or by the allosteric model of DiCera (Di Cera, E., C. H. Robert, and S. J. Gill. 1987. Biochemistry.26:4003-4008).
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