High-resolution proton nuclear magnetic resonance studies of sickle cell hemoglobin.

High-resoluiton proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate sickle cell hemoglobin. The hyperfine shifted, the ring-current shifted, and the exchangeable proton resonances suggest that the heme environment and the subunit interfaces of the sickle cell hemoglobin molecule are normal. These results suggest that the low oxygen affinity in sickle cell blood is not due to conformational alterations in the heme environment or the subunit interfaces. The C-2 proton resonances of certain histidyl residues can serve as structural probes for the surface conformation of the hemoglobin molecule. Several sharp resonances in sickle cell hemoglobin are shifted upfield from their positions in normal adult hemoglobin. These upfield shifts, which are observed in both oxy and deoxy forms of the molecule under various experimental conditions, suggest that some of the surface residues of sickle cell hemoglobin are altered and they may be in a more hydrophobic environment as compared with that of normal human adult hemoglobin. These differences in surface conformation are pH and ionic strength specific. In particular, upon the addition of organic phosphates to normal and sickle cell hemoglobin samples, the differences in their aromatic proton resonances diminish. These changes in the surface conformation may, in part, be responsible for the abnormal properties of sickle cell hemoglobin.     

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

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...     

High-resolution proton nuclear magnetic resonance studies of human gastrin

High-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz has been used to study the behavior of human gastrin in aqueous solution. A large number of resonances have been assigned by analysis of one- and two-dimensional NMR spectra and the effects of pH and by comparison with the spectrum of des-less than Glu1-gastrin. In gastrin, the ratio of cis to trans conformations around the Gly-2 to Pro-3 peptide bond is 3:7. This is reflected in splitting of the resonances of several neighboring residues and of a residue distant in the sequence, Tyr-12. The pKa of Tyr-12 is 10.7. Sulfation of this residue perturbs the resonances of Tyr-12 and Gly-13 but has very little effect on the rest of the spectrum. A study of the temperature dependence shows that several perturbed resonances move toward their expected positions as the temperature is raised but with a linear dependence on temperature, consistent with a redistribution of populations among accessible local conformations rather than a cooperative conformational change. Addition of Na+ or Ca2+ causes only minor changes in the spectrum. The paramagnetic metal ion Co2+ produces a number of spectral changes, reflecting strong binding to at least one site involving the Glu residues and weaker binding to Asp-16.     

Proton magnetic resonance studies of human adult hemoglobin in water

The preferential binding of oxygen to the α hemes of normal human adult hemoglobin in the presence of organic phosphates in D₂O at neutral pD has been confirmed in H₂O. Additionally, it has been found that the preferential binding of oxygen is pH dependent with random ligation being observed at pH ~ 9.     

The alkylation of hemoglobin S by nitrogen mustard. High resolution proton nuclear magnetic resonance studies.

Sickle cell hemoglobin (Hb S) treated with nitrogen mustard (bis(beta-chloroethyl)methylamine hydrochloride) gives two reaction products, one labile and one stable. After dialysis against buffer solution, the remaining stable product is found to inhibit the polymerization of deoxyhemoglobin S. High resolution proton nuclear magnetic resonance has been used to study the structure and function of this stable product and to investigate the nature of the binding sites of nitrogen mustard to the hemoglobin molecule. The NMR results suggest that the nitrogen mustard treatment of Hb S does not alter the heme environment or the subunit interfaces of the hemoglobin molecule. Moreover, the NMR spectra have also shown that the nitrogen mustard reacts with the beta2 histidines of the hemoglobin molecule and have suggested that several other surface amino acid residues of the hemoglobin molecule are also affected by the nitrogen mustard alkylation. These NMR findings are in good agreement with the data obtained from biochemical studies of nitrogen mustard-treated Hb S. The NMR spectra also indicate that nornitrogen mustard (which is also effective in inhibiting sickling) binds with the hemoglobin molecule in a manner identical with nitrogen mustard. Sulfur mustard, on the other hand, produces no observable changes in the aromatic proton resonances, which is consistent with the fact that it does not inhibit the polymerization of deoxy-Hb S.     

Nuclear magnetic resonance studies of the solution conformation of nucleoside diphosphohexoses and their components.

The solution conformations of UDPG, UDPGN, UDPGal, UDPM, UDPGluc, UDPGalc, ADPG, ADPM, GDPG, GDPM, and CDPG and their components Glu-1-P, Gal-1-P, Man-1-P, Gluc-1-P, Galc-1-P, ADP, GDP, UDP, and CDP are studied by high resolution fast Fourier transform nuclear magnetic resonance spectroscopy with iterative computer line shape simulation. The following results were observed. (1) The six-membered ring is in 4C1 chair form with the C(5')-C(6') bond in gg equilibrium tg equilibrium for the derivatives of glucose and mannose and gt equilibrium tg for those of galactose. (2) No conformational preference can be detected for C(1')-O(1') bond in hexose-1'-P moiety. (3) Chemical shift dependencies for the pyranoid ring protons and their structural and conformational relations are: (a) axial proton is at higher field than equatorial: (b) the shielding effect of a gauche vicinal hydroxyl group is stronger than a trans vicinal; (c) the vicinity of a hydroxyl group located more than three bonds away tends to shift the proton downfield. (4) The conformation of the nucleoside 5'-diphosphate part is [anti, 2E equilibrium 3E, g'g' equilibrium g't', g'g' equilibrium g'/t'], with slight variation of each conformation occuring for individual compounds. (5) No significant interactions are detected between the hexose and nucleoside parts in the nucleoside diphosphohexoses, and the hexose and nucleoside components display the same conformational preference as they become integrated to form nucleoside diphosphohexoses.     

Nuclear magnetic resonance studies of amphiphile hydration.

The nuclear magnetic resonance signal of water which remains unfrozen at ?25 °C in the presence of phosphatidylcholine has been used to determine the hydration of this amphiphile. The effects of cholesterol and sodium dodecylsulfate on both the area and linewidth of this signal indicate that these molecules cause significant changes in the structure of phosphatidylcholine vesicles in solution. Studies on other amphiphiles indicate that, whereas phosphatidylethanolamine has a hydration similar to phosphatidylcholine, species with just one hydrocarbon chain such as sodium dodecylsulfate and dodecyltrimethylammonium bromide have little, if any, hydration when assayed via the nuclear magnetic resonance experiment.     

Nuclear magnetic resonance studies of the denaturation of ubiquitin.

The effects of pH, temperature and guanidine hydrochloride concentration on the structure of ubiquitin, a polypeptide which can activate adenylate cyclase and can mimic thymopoietin induced differentiation of prothymocytes, were monitored using nuclear magnetic resonance spectroscopy. This relatively small polypeptide (molecular weight of 8541) exhibits a remarkable stability towards pH and temperature changes. At 7 M guanidine hydrochloride concentration, the structure of ubiquitin is essentially a random coil.