Abstract
Oxygen equilibrium curves of the extracellular hemoglobin from Lumbricus terrestris were determined under a variety of conditions. These data were characterized by (i) a rather small free energy of cooperativity (1.6-2.8 kcal/mol), (ii) a large and strongly pH-dependent Hill coefficient with a maximum value of 7.9, (iii) a high sensitivity of the upper asymptote of the Hill plot to pH, and (iv) a maximum association constant as large as that of the free beta subunit of human hemoglobin A. The effects of LiCl, KCl, NaCl, BaCl2, CaCl2, SrCl2, and MgCl2 on the oxygen equilibrium were measured. Cations, not Cl-, were found to control oxygen binding. Divalent cations have a larger effect on oxygen affinity than monovalent cations, and their effectiveness decreased in the order listed above within each valence class. These specific effects depend in part on ionic radius and cannot be explained in terms of ionic strength. The data indicate that the oxygenation-linked binding of a Ca2+ ion is accompanied by the release of two protons; the binding of a Na+ ion is associated with the release of one proton. These findings indicate that the oxygenation-linked cation-binding site contains two acid groups that do not readily dissociate their protons except when replaced by cations. Incubation at either pH 6.2 or 8.9 had no effect on subsequent measurements of oxygen equilibria at pH 7.8. The apparent heat of oxygenation was found to be -11.8, -7.3, and -9.3 kcal/mol at pH 9.0, 7.4, and 6.6, respectively. These differences indicate that proton-binding processes contribute to the heat of oxygenation.
Highlights
Oxygen equilibriumcurves of theextracellular chains of vertebrate hemoglobins [5].The whole moleculehas hemoglobin from Lumbricus terrestris weredetermined under a variety of conditions
Oxygen-binding properties of L. terrestris hemoglobin have been studied by several investigators [7,8,9]
Weber [12] showed that cations control the oxygen affinity of the extracellular hemoglobin of Arenicolu marina. Addition of cations such asNa+ or Mg2+enhanced cooperativity and raised the oxygen affinity by binding to hemoglobin at high levels of oxygenation. He showed that protons lower the oxygen affinity by preferential binding to hemoglobin a t high saturation levels
Summary
10%.The precipitate was redissolved in 0.05 M Tris-HC1,pH 8.0, and Analysis-Oxygenation data were expressed in terms of the Hill twice pelleted a t 40,000 rpm for 2 h. The Monod-Wyman-Changeux (MWC) allosteric model [30] describes the oxygenation of hemoglobin as, and 99% with a variance of 1-2% of P W and n,- values where Pw is the oxygen pressure a t half-saturation and n,- is the maximum value of the Hill coefficient. The ratio of absorbance a t a trough near 505 nm to that ata peak at 576 nm provides an index of methemoglobin The value of this index for hemoglobin solutions between pH 7.0-7.8 before measurements was 2.99 0.03 S.D. Methemoglobin content after oxygen equilibrium measurements was usually estimated to be 6-8% and never more than. Oxygen-binding Properties: Effectof p H and Calcium-Hill plots of oxygen equilibria of L. terrestris hemoglobin are given in Fig. 1.Measurements were made between pH 6.2 and 8.9 under 20 pM (see Fig. 7).
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