ALLOSTERIC EFFECTORS DO NOT ALTER THE OXYGEN-AFFINITY OF HEMOGLOBIN CRYSTALS

In solution, the oxygen affinity of hemoglobin in the T quaternary str ucture is decreased in the presence of allosteric effecters such as pr otons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state h emoglobin in the crystal compared to solution, Rivetti C et al. (1993a , Biochemistry 32:2888-2906) suggested that there are high- and low-af finity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemo globin has the lowest possible oxygen affinity because the salt bridge s remain intact upon oxygenation. Binding of allosteric effecters in t he 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 quate rnary structure in the presence of the ''strong'' allosteric effecters , inositol hexaphosphate and bezafibrate. In solution, these effecters reduce the oxygen affinity of the T state by 10-30-fold. We find no c hange in affinity (< 10%) of the crystal. The crystal binding curve, m oreover, is noncooperative, which is consistent with the essential fea ture of the two-state allosteric model of Monod J, Wyman J, and Change ux 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 s ubunits. This was shown by calculating the separate alpha and beta sub unit binding curves from the two sets of polarized optical spectra usi ng geometric factors from the X-ray structures of deoxygenated and ful ly oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).