Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

Interactions of nicotinamide adenine dinucleotides with varied states and forms of hemoglobin

Spectrofluorometric techniques were used to quantify NADPH-hemoglobin interactions based on the quenching of NADPH fluorescence upon binding to hemoglobin. Fluorometric titrations were carried out with hemoglobin in varied states and with hemoglobins in which the beta-chain anion site is altered. At pH 6.5 in 0.05 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, NADPH binds with high affinity, Kd = 1.03 microM, to deoxy human hemoglobin tetramers. Lower affinity binding of NADPH occurs as the beta-chain anion-binding site is discharged by increasing the pH. Moreover, the cofactor binds in a 1:1 ratio to deoxy tetramers, inositol hexaphosphate binds competitively, and binding is decreased in hemoglobins whose structural alterations result in decreased effects of 2,3-diphosphoglycerate. The cofactor binds to oxidized (met) hemoglobin with an estimated Kd of 33.3 microM but has little or no affinity for the oxy form. These results indicate that NADPH binds at the beta-chain anion-binding site and can be considered as a fluorescent analog of 2,3-diphosphoglycerate. Fluorescence measurements gave no indication of NADPH binding to deoxygenated ferrous or ferric myoglobin. Reductive processes within the erythrocyte, such as reduction of met hemoglobin and hemoglobin-catalyzed enzymatic reactions, may be affected by the significant binding of the reduced cofactor to both deoxygenated and oxidized hemoglobin. Cofactor-hemoglobin interactions predict a shift in redox potential as red cells become oxygenated, which may account for unexplained oxygen-linked shifts in red cell metabolism.     

pH-dependent Soret difference spectra of the deoxy and carbonmonoxy forms of human hemoglobin and its derivatives.

The transition from deoxy to oxystructure of hemoglobin A (Hb) is accompanied by the breaking of the salt bridges formed by C-terminal residues in deoxy-Hb. This, in turn, changes the state of the heme. The switch between these different allosteric forms can be followed by changes in the optical absorbance spectra (Perutz, M. F., Ladner, J. E., Simon, S. R., and Ho, C. (1974), Biochemistry 13, 2163). Using difference spectroscopy in the soret region, pH-dependent spectral changes of Hb and its derivatives (carbamylated at both the alpha-NH2 groups, alpha2cbeta2c; N-ethylsuccinimide hemoglobin, NES-Hb) in their deoxy and carbonmonoxy forms were measured. From these measurements, the pK values of histidine-146beta and valine-1alpha in deoxy-Hb were determined to be 8.6 +/- 0.2 and 7.7 +/- 0.1, respectively. In carbonmonoxy-Hb a pK value of 6.3 +/- 0.1 was found.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Muscle oxygenation and pulmonary gas exchange kinetics during cycling exercise on-transitions in humans.

Near-infrared spectroscopy (NIRS) was utilized to gain insights into the kinetics of oxidative metabolism during exercise transitions. Ten untrained young men were tested on a cycle ergometer during transitions from unloaded pedaling to 5 min of constant-load exercise below (VT) the ventilatory threshold. Vastus lateralis oxygenation was determined by NIRS, and pulmonary O2 uptake (Vo --> Vo2) was determined breath-by-breath. Changes in deoxygenated hemoglobin + myoglobin concentration Delta[deoxy(Hb + Mb)] were taken as a muscle oxygenation index. At the transition, [Delta[deoxy(Hb + Mb)]] was unmodified [time delay (TD)] for 8.9 +/- 0.5 s at VT (both significantly different from 0) and then increased, following a monoexponential function [time constant (tau) = 8.5 +/- 0.9 s for VT]. For >VT a slow component of Delta[deoxy(Hb + Mb)] on-kinetics was observed in 9 of 10 subjects after 75.0 +/- 14.0 s of exercise. A significant correlation was described between the mean response time (MRT = TD + tau) of the primary component of Delta[deoxy(Hb + Mb)] on-kinetics and the tau of the primary component of the pulmonary Vo2 on-kinetics. The constant muscle oxygenation during the initial phase of the on-transition indicates a tight coupling between increases in O2 delivery and O2 utilization. The lack of a drop in muscle oxygenation at the transition suggests adequacy of O2 availability in relation to needs.     

Metal complexes as allosteric effectors of human hemoglobin: an NMR study of the interaction of the gadolinium(III) bis(m-boroxyphenylamide)diethylenetriaminepentaacetic acid complex with human oxygenated and deoxygenated hemoglobin

The boronic functionalities on the outer surface of the Gd(III) bis(m-boroxyphenylamide)DTPA complex (Gd(III)L) enable it to bind to fructosamine residues of oxygenated glycated human adult hemoglobin. The formation of the macromolecular adduct can be assessed by NMR spectroscopy via observation of the enhancement of the solvent water proton relaxation rate. Unexpectedly, a strong binding interaction was also observed for the oxygenated unglycated human adult hemoglobin, eventually displaying a much higher relaxation enhancement. From relaxation rate measurements it was found that two Gd(III)L complexes interact with one hemoglobin tetramer (KD = 1.0 x 10(-5) M and 4.6 x 10(-4) M, respectively), whereas no interaction has been observed with monomeric hemoproteins. A markedly higher affinity of the Gd(III)L complex has been observed for oxygenated and aquo-met human adult hemoglobin derivatives with respect to the corresponding deoxy derivative. Upon binding, a net change in the quaternary structure of hemoglobin has been assessed by monitoring the changes in the high-resolution 1H-NMR spectrum of the protein as well as in the Soret absorption band. On the basis of these observations and the 11B NMR results obtained with the diamagnetic La(III)L complex, we suggest that the interaction between the lanthanide complex and deoxygenated, oxygenated, and aquo-met derivatives of human adult hemoglobin takes place at the 2, 3-diphosphoglycerate (DPG) binding site, through the formation of N-->B coordinative bonds at His143beta and His2beta residues of different beta-chains. The stronger binding to the oxygenated form is then responsible for a shift of the allosteric equilibrium toward the high-affinity R-state. Accordingly, Gd(III)L affinity for oxygenated human fetal hemoglobin (lacking His143beta) is significantly lower than that observed for the unglycated human adult tetramer.     

Water proton magnetic resonance studies of normal and sickle erythrocytes. Temperature and volume dependence.

The temperature and cell volume dependence of the NMR water proton line-width, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T2) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T1) shows no significant change upon deoxygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T1 and T2 as the cell hydration is changed indicate that these parameters are affected only slightly by a 10-20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T1 for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the "bound" water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at -15 degrees C to 500 Hz at -36 degrees C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T1 data go through a minimum at -35 degrees C for measurements at 44.4 MHz and -50 degrees C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irrotationally bound water, is altered during the sickling process.     

Simultaneous determination of low free Mg2+ and pH in human sickle cells using 31P NMR spectroscopy

The concentrations of free magnesium, [Mg(2+)](free), [H(+)], and [ATP] are important in the dehydration of red blood cells from patients with sickle cell anemia, but they are not easily measured. Consequently, we have developed a rapid, noninvasive NMR spectroscopic method using the phosphorus chemical shifts of ATP and 2,3-diphosphoglycerate (DPG) to determine [Mg(2+)](free) and pH(i) simultaneously in fully oxygenated whole blood. The method employs theoretical equations expressing the observed chemical shift as a function of pH, K(+), and [Mg(2+)](free), over a pH range of 5.75-8.5 and [Mg(2+)](free) range 0-5 mm. The equations were adjusted to allow for the binding of hemoglobin to ATP and DPG, which required knowledge of the intracellular concentrations of ATP, DPG, K(+), and hemoglobin. Normal oxygenated whole blood (n = 33) had a pH(i) of 7.20 +/- 0.02, a [Mg(2+)](free) of 0.41 +/- 0.03 mm, and [DPG] of 7.69 +/- 0.47 mm. Under the same conditions, whole sickle blood (n = 9) had normal [ATP] but significantly lower pH(i) (7.10 +/- 0.03) and [Mg(2+)](free) (0.32 +/- 0.05 mm) than normal red cells, whereas [DPG] (10.8 +/- 1.2 mm) was significantly higher. Because total magnesium was normal in sickle cells, the lower [Mg(2+)](free) could be attributed to increased [DPG] and therefore greater magnesium binding capacity of sickle cells.     

Determination of glucose oxidase oxidation-reduction potentials and the oxygen reactivity of fully reduced and semiquinoid forms.

The oxidation-reduction potential values for the two electron transfers to glucose oxidase were obtained at pH 5.3, where the neutral radical is the stable form, and at pH 9.3, where the anion radical is the stable form. The midpoint potentials at 25 degrees were: pH 5.3 EFl1ox + e- H+ equilibrium EFlH. Em1 = -0.063 +/- 0.011 V EFlH. + e- + H+ equilibrium EFlredH2 Em2 = -0.065 +/- 0.007 V pH 9.3 EFlox + e- EFi- Em1 = -0.200 +/- 0.010 V EFi- + e- + H+ equilibrium EFlredH- Em2 = -0.240 +/- 0.005 V All potentials were measured versus the standard hydrogen electrode (SHE). The potentials indicated that glucose oxidase radicals are stabilized by kinetic factors and not by thermodynamic energy barriers. The pK for the glucose oxidase radical was 7.28 from dead time stopped flow measurements and the extinction coefficient of the neutral semiquinone was 4140 M-1 cm-1 at 570 nm. Both radical forms reacted with oxygen in a second order fashion. The rate at 25 degrees for the neutral semiquinone was 1.4 X 10(4) M-1 s-1; that for the anion radical was 3.5 X 10(4) M-1 s-1. The rate of oxidation of the neutral radical changed by a factor of 9 for a temperature difference of 22 degrees. For the anion radical, the oxidation rate changed by a factor of 6 for a 22 degrees change in temperature. We studied the oxygen reactivity of the 2-electron reduced form of the enzyme over a wide wavelength range and failed to detect either oxygenated flavin derivatives or semiquinoid forms as intermediates. The rate of reoxidation of fully reduced glucose oxidase at pH 9.3 was dependent on ionic strength.     

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on cobalt myoglobins and hemoglobins. Interaction of sperm whale myoglobin and Glycera hemoglobin with molecular oxygen.

The pH dependence of the electron paramagnetic resonance (EPR) spectrum and oxygen affinity of cobaltous porphyrin-containing myoglobin (CoMb) have been examined. The hyperfine structures of the EPR spectrum of oxy-CoMb undergo small, reversible pH-dependent changes with pK values of 5.33, 5.55, and 5.25 +/- 0.05 for proto-, meso-, and deutero-CoMb's, respectively, whereas deoxy-CoMb does not exhibit any pH dependence of its EPR spectrum. The partial pressure of oxygen at half-saturation of proto-CoMb decreases from 26 to 42 Torr on lowering the pH from 7.0 to 4.8. For comparison, we have prepared cobaltous porphyrin-containing monomeric Glycera hemoglobin (CoHb (Glycera)), in which the distal histidyl group of myoglobin is replaced by a leucyl residue, and examined the equilibria and kinetics of its oxygenation and EPR spectrum. CoHb (Glycera) has exhibited a very low oxygen affinity (p50 = 7 X 10(2) Torr at 5 degrees) and a large dissociation rate constant (more than 8 X 10(4) S-1 at 5 degrees). The EPR spectrum of oxy-CoHb (Glycera) was affected by neither pH nor replacement of H2O with D2O. Low temperature photodissociation studies by EPR and spectrophotometry have shown that the photolyzed form of the ligated hemoglobin (Glycera) is similar to its deoxy form, in contrast to myoglobin which gives a new intermediate states as the photolyzed form. These differences between CoMb and CoHb (Glycera) are interpreted with relation to the possible role of the distal histidyl residue in CoMb.     

Glutathione mixed disulfides and heterogeneity of chicken hemoglobins.

1. Adult chicken hemoglobins Hb A and Hb D interact with glutathione disulfide, GSSG. The major hemoglobin, Hb A, forms at least two new components, termed GHb AI and GHb AII, and Hb D forms at least one, GHb DI. 2. At pH 8.0 and 5 degrees C, glutathione disulfide (GSSG) in a molar excess of 50 x took 6 days to complete the reaction, although at pH 8.6 and 41 degrees C only 1 hr was needed, where the hemoglobins Hb A and Hb D were converted to their most mobile forms GHb AII and GHb DI. 3. Slight molar excess (2.7 GSSG/Hb, pH 7.4, 41 degrees C), reacting for 1 hr, showed extensive formation of GHb AI and some GHb AII. 4. Electrophoretic patterns, from the reaction products of 54 GSSG/Hb excess at different times, showed a marked pH dependence. 5. Titration with pCMB (p-chloromercuribezoic acid) of DTE (dithioerythrytol)-reduced samples showed 8.0 +/- 0.4 (N = 5) -SH (sulfhydryl) per tetramer. In hemolysates not reacted with DTE, 6.0 +/- 0.4 (N = 3) -SH were detected. 6. DTE-reduced and GSSG-reacted hemoglobins showed 4.6 +/- 0.5 (N = 7) -SH and 1.5 +/- 0.4 (N = 6) -SH, respectively, as titrated by DTNB, pH 8.0. DTE-reduced hemoglobins showed four fast-reacting -SH groups, no longer present in GSSG-reacted hemoglobins. 7. Our data indicate that chicken GHb AI and GHb DI probably have two glutathionyl residues per tetramer whereas GHb AII has four.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of differences in optical properties of intermediate oxygenated species of hemoglobin A0 on Adair constant determination

Careful evaluation of the so-called isosbestic properties of oxygenated and deoxygenated hemoglobin spectra demonstrates that the spectral changes are not strictly linear with respect to the degree of saturation. In order to quantify the extent of nonlinearity, optical measurements of O2 binding to human hemoglobin were made at different wavelengths in the Soret region approaching the presumed isosbestic point. The results indicate that the extinction coefficient of intermediate oxygenated hemoglobin is 1% less than that of the fully oxygenated hemoglobin, with a resulting 3% (+/- 0.15%) nonlinearity effect on measurements taken at the peak of the oxygenated hemoglobin spectrum (414 nm). The lack of isosbestic conditions allows one to investigate the functional properties of the oxygenated intermediates directly. The small difference in the absorbance of different oxygenated species has practically no influence on the determination of Adair constants at wavelengths removed from the critical isosbestic region.     

Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins

A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, base-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol(-1), n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer-dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (deltaGc)--the energy barrier between the allosteric transition from deoxy to oxy hemoglobin.     

Hagfish hemoglobins: structure, function, and oxygen-linked association

Cyclostomes, hagfishes and lampreys, contain hemoglobins that are monomeric when oxygenated and polymerize to dimers or tetramers when deoxygenated. The three major hemoglobin components (HbI, HbII, and HbIII) from the hagfish Myxine glutinosa have been characterized and compared with lamprey Petromyzon marinus HbV, whose x-ray crystal structure has been solved in the deoxygenated, dimeric state (Heaslet, H. A., and Royer, W. E., Jr. (1999) Structure 7, 517-526). Of these three, HbII bears the highest sequence similarity to P. marinus HbV. In HbI and HbIII the distal histidine is substituted by a glutamine residue and additional substitutions occur in residues located at the deoxy dimer interface of P. marinus HbV. Infrared spectroscopy of the CO derivatives, used to probe the distal pocket fine structure, brings out a correlation between the CO stretching frequencies and the rates of CO combination. Ultracentrifugation studies show that HbI and HbIII are monomeric in both the oxygenated and deoxygenated states under all conditions studied, whereas deoxy HbII forms dimers at acidic pH values, like P. marinus HbV. Accordingly, the oxygen affinities of HbI and HbIII are independent of pH, whereas HbII displays a Bohr effect below pH 7.2. HbII also forms heterodimers with HbIII and heterotetramers with HbI. The functional counterparts of heteropolymer formation are cooperativity in oxygen binding and the oxygen-linked binding of protons and bicarbonate. The observed effects are explained on the basis of the x-ray structure of P. marinus HbV and the association behavior of site-specific mutants (Qiu, Y., Maillett, D. H., Knapp, J., Olson, J. S., and Riggs, A. F. (2000) J. Biol. Chem. 275, 13517-13528).     

Nonlinear optical effects in oxygen-binding reactions of hemoglobin A0

Optical spectra have been taken in the Soret band (440-400 nm) under different oxygen partial pressures for hemoglobin (Hb) A0 at pH 7.0, 15 degrees C, 2-3 mM heme, 30 mM inositol hexaphosphate, 0.1 Hepes and 0.1 M NaCl. Application of the matrix method of singular value decomposition (SVD) to the difference spectra for different oxygen pressures shows the presence of at least two distinct optical transitions. From this result one concludes that the optical response to oxygen binding is nonlinear in the Soret band. The degree of nonlinearity has been determined by fitting the data at different wavelengths to the four-step reaction Adair equation with the inclusion of optical parameters that describe the intermediate oxygenated species. It is found that the data are well-represented by two optical parameters at each wavelengths, one which represents the optical change for the addition of the first and second oxygen molecules and the other which corresponds to the change for the addition of the third and fourth oxygen molecules. The ratio of these optical parameters depends only moderately upon wavelength with an average value of 0.8 over the Soret band. Thus, there is an approx. 20% smaller optical response for the first two ligated species than that for the last two ligated species. The overall Adair equilibrium constants are evaluated as follows: beta 1 = 0.081 +/- 0.003 Torr-1, beta 2 = 2.53 x 10(-3) +/- 2.4 x 10(-4) Torr-2, beta 3 = 1.25 x 10(-5) +/- 1.0 x 10(-6) Torr-3, beta 4 = 1.77 x 10(-6) +/- 1.5 x 10(-7) Torr-4.     

Hydrogen-tritium exchange survey of allosteric effects in hemoglobin

The oxy and deoxy forms of hemoglobin display major differences in H-exchange behavior. Hydrogen-tritium exchange experiments on hemoglobin were performed in the low-resolution mode to observe the dependence of these differences on pH (Bohr effect), organic phosphates, and salt. Unlike a prior report, increasing pH was found to decrease the oxy-deoxy difference monotonically, in general accordance with the alkaline Bohr effect. A prior report that the H-exchange difference between oxy- and deoxyhemoglobin vanishes at pH 9, and thus appears to reflect the Bohr effect alone, was found to be due to the borate buffer used, which at high pH tends to abolish the oxy-deoxy difference in a limited region of the H-exchange curve. Effects on hemoglobin H exchange due to organic phosphates parallel the differential binding of these agents (inositol hexaphosphate more than diphosphoglycerate, deoxy more than oxy, at low pH more than at high pH). Added salt slows H exchange of deoxyhemoglobin and has no effect on the oxy form. These results display the sensitivity of simple H-exchange measurements for finding and characterizing effects on structure and dynamics that may occur anywhere in the protein and help to define conditions for higher resolution approaches that can localize the changes observed.     

Mechanism of inhibition of sickling by dimethyl adipimidate. Effects of intertetramer cross-linking

Dimethyl adipimidate (DMA), an effective antisickling agent in vitro, reacts with free amino groups producing chemically modified and cross-linked molecules. In this report, we have investigated the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization. Since the extent of cross-linking is pH-dependent, we first compared the solubilities of deoxygenated hemolysates prepared from sickle cells previously treated with dimethyl adipimidate at either pH 7.4 or 8.4. The solubility of the hemolysate increased from 18.6 +/- 0.8 g/dl in the untreated sample to 20.9 +/- 1.5 g/dl (pH 7.4) and to 25.4 +/- 3.0 g/dl (pH 8.4) after dimethyl adipimidate treatment. Removal of cross-linked hemoglobin tetramers from hemolysate obtained from dimethyl adipimidate-treated cells abolished part of this effect; at pH 7.4, the solubility decreased from 20.9 +/- 1.5 to 19.4 +/- 0.2 and at pH 8.4 from 25.4 +/- 3.0 to 21.0 +/- 1.5. However, the ratio of [14C]DMA-labelled hemoglobin in the sol phase to that in the gel phase in the unfractionated hemolysate was 1.17 at pH 7.4 and 1.25 at pH 8.4, suggesting that part of the cross-linked hemoglobin tetramers was incorporated into the gel. In order to further investigate the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization, we separated cross-linked hemoglobin tetramers on a gel-filtration column, prepared mixtures of untreated sickle hemoglobin and cross-linked hemoglobin tetramers and studied the polymerization of these mixtures. The Csat of the untreated hemolysate increased progressively from 18.6 +/- 0.8 to 22.5 +/- 0.8 g/dl with 33% cross-linked hemoglobin tetramers. The hemoglobin concentration in the gel decreased from 43 +/- 1.0 to 33.8 +/- 1.0 g/dl with 33% cross-linked hemoglobin tetramers, while the pellet volume fraction, phi p, increased with and almost approached 1 at 50% cross-linked hemoglobin tetramers. In addition, the sol phase contained a higher molecular weight distribution of cross-linked hemoglobin tetramers than the gel phase. These observations suggest that a loose polymer was formed in the gel phase with a hemoglobin concentration much lower than that of the control. Thus, polymerization of sickle hemoglobin is inhibited by: (1) exclusion of higher molecular weight cross-linked hemoglobin tetramers from the gel, and (2) loose incorporation of cross-linked hemoglobin tetramers into the gel, perhaps preventing lateral packing and formation of tightly ordered fibers.     

Coupling of the oxygen-linked interaction energy for inositol hexakisphosphate and bezafibrate binding to human HbA0

The energetics of signal propagation between different functional domains (i.e. the binding sites for O2, inositol hexakisphospate (IHP), and bezafibrate (BZF)) of human HbA0 was analyzed at different heme ligation states and through the use of a stable, partially heme ligated intermediate. Present data allow three main conclusions to be drawn, and namely: (i) IHP and BZF enhance each others binding as the oxygenation proceeds, the coupling free energy going from close to zero in the deoxy state to -3.4 kJ/mol in the oxygenated form; (ii) the simultaneous presence of IHP and BZF stabilizes the hemoglobin T quaternary structure at very low O2 pressures, but as oxygenation proceeds it does not impair the transition toward the R structure, which indeed occurs also under these conditions; (iii) under room air pressure (i.e. pO2 = 150 torr), IHP and BZF together induce the formation of an asymmetric dioxygenated hemoglobin tetramer, whose features appear reminiscent of those suggested for transition state species (i.e. T- and R-like tertiary conformation(s) within a quaternary R-like structure).     

Mechanistic studies of the oxidation of pyridoxalated hemoglobin polyoxyethylene conjugate by nitrogen monoxide

Pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), a modified human-derived hemoglobin, is currently in clinical trials as a nitrogen monoxide scavenger for the treatment of shock. Stopped-flow spectroscopy studies of the reaction between nitrogen monoxide and PHP indicate that at pH 7 the second-order rate constant is (88 +/- 3) x 10(6) M(-1) s(-1), a value very similar to that for the unmodified human hemoglobin. At alkaline pH the reaction proceeds via the intermediate peroxynitrito complex PHP-Fe(III)OONO, which rapidly decomposes to nitrate and the iron(III) form of PHP. The rate of decay of PHP-Fe(III)OONO increases significantly with decreasing pH such that it does not accumulate in concentrations large enough to be observed spectroscopically under neutral or acidic conditions. Ion chromatographic analysis of the nitrogen-containing products of the NO(*)-mediated reaction of PHP shows that nitrate is formed quantitatively at both pH 7 and pH 9.