Protein Science, Vol 6, Issue 2 484-489, Copyright © 1997 by Cold Spring Harbor Laboratory Press

FOR THE RECORD

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals

A. MOZZARELLI, C. RIVETTI, G. L. ROSSI, W. A. EATON and E. R. HENRY
Institute of Biochemical Sciences, University of Parma, 43100 Parma, Italy

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the ``strong'' allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (<10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the {alpha} and {beta} subunits. This was shown by calculating the separate {alpha} and {beta} subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).
CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L. Ronda, S. Bruno, C. Viappiani, S. Abbruzzetti, A. Mozzarelli, K. C. Lowe, and S. Bettati Circular dichroism spectroscopy of tertiary and quaternary conformations of human hemoglobin entrapped in wet silica gels Protein Sci., August 1, 2006; 15(8): 1961 - 1967. [Abstract] [Full Text] [PDF]

				C. Viappiani, S. Bettati, S. Bruno, L. Ronda, S. Abbruzzetti, A. Mozzarelli, and W. A. Eaton New insights into allosteric mechanisms from trapping unstable protein conformations in silica gels PNAS, October 5, 2004; 101(40): 14414 - 14419. [Abstract] [Full Text] [PDF]

				R. W. Noble, L. D. Kwiatkowski, H. L. Hui, S. Bruno, S. Bettati, and A. Mozzarelli Correlation of protein functional properties in the crystal and in solution: The case study of T-state hemoglobin Protein Sci., July 1, 2002; 11(7): 1845 - 1849. [Abstract] [Full Text] [PDF]

				S. Bruno, M. Bonaccio, S. Bettati, C. Rivetti, C. Viappiani, S. Abbruzzetti, and A. Mozzarelli High and low oxygen affinity conformations of T state hemoglobin Protein Sci., November 1, 2001; 10(11): 2401 - 2407. [Abstract] [Full Text] [PDF]

				J. S. Kavanaugh, J. A. Weydert, P. H. Rogers, A. Arnone, H. L. Hui, A. M. Wierzba, L. D. Kwiatkowski, P. Paily, R. W. Noble, S. Bruno, et al. Site-directed mutations of human hemoglobin at residue 35{beta}: A residue at the intersection of the {alpha}1{beta}1, {alpha}1{beta}2, and {alpha}1{alpha}2 interfaces Protein Sci., September 1, 2001; 10(9): 1847 - 1855. [Abstract] [Full Text] [PDF]

				S. Bettati, L. D. Kwiatkowski, J. S. Kavanaugh, A. Mozzarelli, A. Arnone, G. L. Rossi, and R. W. Noble Structure and Oxygen Affinity of Crystalline des-His-146beta Human Hemoglobin in the T State J. Biol. Chem., December 26, 1997; 272(52): 33077 - 33084. [Abstract] [Full Text] [PDF]

				S. Bettati and A. Mozzarelli T State Hemoglobin Binds Oxygen Noncooperatively with Allosteric Effects of Protons, Inositol Hexaphosphate, and Chloride J. Biol. Chem., December 19, 1997; 272(51): 32050 - 32055. [Abstract] [Full Text] [PDF]