Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide

Abstract

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...

Interfacial and Distal-Heme Pocket Mutations Exhibit Additive Effects on the Structure and Function of Hemoglobin

“…Significant subunit heterogeneity is expected to reduce the apparent cooperativity as measured by n 50 (60, 61). NMR studies have detected subunit heterogeneity of Hb A for the first step of O 2 binding in the presence of organic phosphate (62). Table 2 shows that reductions in the n 50 values are observed for rHbs containing αL29F and αL29W substitutions, as would be expected if the α-subunit has much higher and much lower O 2 affinity, respectively, than its partner β-subunit.…”

“…Thus, for Hb in RBCs, a P 50 of 20 to 30 Torr is optimal and most workers feel that the same logic applies to extracellular HBOCs. However, this view has been challenged (62). Extracellular HBOCs have no unstirred layers surrounding them, permeate the cell-free plasma layer lining the vessel walls of arteries and arterioles, and as result, deliver oxygen two to three times more efficiently on an iron basis than RBCs alone (23).…”

Structure-specific model of hemoglobin cooperativity.

“…Spectroscopic studies of probes sensitive to specific changes are providing valuable information for testing models of cooperativity; an example is the attempt to distinguish between aand (3chain oxygenation by NMR (14,15) and by optical measurements (16). Highly accurate equilibrium measurements for oxygen binding in hemoglobin have been made under carefully controlled conditions (17,18); this removes some of the uncertainties present in the Roughton-Lyster data (19) on which the original fits were based.…”

A quantitative model for the cooperative mechanism of human hemoglobin.

“…1-4. A number of these can be tested-e.g., by extended NMR studies of the fractional saturation of a and (3chains (34,35) and the fractional quaternary structure change (33,34). Within the framework provided by this model, a more rigorous set of rules may be incorporated for the pairwise subunit interactions.…”

“…(ii) We constrained the ratio of probabilities of binding oxygen by the a and 8 chains to be 1.0 ± 0.05 in the range between zero and half-saturation of tetramers, in accord with the results of NMR determinations (34,35). xiii) At pH 7.4 the change in quaternary structure with fractional saturation of tetramers Y4 was constrained to be linear to within 5% between Y4 = 0 and Y4 = 0.5, in accord with results of NMR determinations (34).…”