Binding of inositol hexaphosphate to human methemoglobin.

The observed static difference spectrum produced by inositol hexaphosphate binding to methemoglobin is the sum of a very fast and a slow spectral transition. The more rapid absorbance change is too fast to be measured by stopped flow techniques, whereas the slow change exhibits a half-time in the range 1 to 6 s. From the pH dependence of the rapidly formed difference spectrum and from a series of heme ligand binding studies, the rapid phase is interpreted to reflect a localized tertiary conformational change which immediately accompanies inositol hexaphosphate binding and results in a selective increase in spin and reactivity of the beta chain heme groups. In contrast, the slow phase appears to reflect a first order isomerization process which involves only a small portion (less than 10%) of the hemoglobin molecules and results primarily in a marked alteration of the spectral properties of the alpha chains with little change in spin. While the rapid spectral transition cannot be directly related to the overall quaternary transition which occurs during oxygen binding to ferrous deoxyhemoglobin, the slow spectral transition may represent the abortive formation of a deoxyhemoglobin A-like conformation which is inhibited in both rate and extent by the presence of water molecules bound to the heme iron atoms.     

A pH-dependent aquomet-to-hemichrome transition in crystalline horse methemoglobin

In 1947, Perutz and co-workers reported that crystalline horse methemoglobin undergoes a large lattice transition as the pH is decreased from 7.1 to 5.4. We have determined the pH 7.1 and 5.4 crystal structures of horse methemoglobin at 1.6 and 2.1 A resolution, respectively, and find that this lattice transition involves a 23 A translation of adjacent hemoglobin tetramers as well as changes in alpha heme ligation and the tertiary structure of the alpha subunits. Specifically, when the pH is lowered from 7.1 to 5.4, the Fe(3+) alpha heme groups (but not the beta heme groups) are converted from the aquomet form, in which the proximal histidine [His87(F8)alpha] and a water molecule are the axial heme ligands, to the hemichrome (bishistidine) form, in which the proximal histidine and the distal histidine [His58(E7)alpha] are the axial heme ligands. Hemichrome formation is coupled to a large tertiary structure transition in the eight-residue segment Pro44(CD2)alpha-Gly51(D7)alpha that converts from an extended loop structure at pH 7.1 to a pi-like helix at pH 5.4. The formation of the pi helix forces Phe46(CD4)alpha out of the alpha heme pocket and into the interface between adjacent hemoglobin tetramers where it participates in crystal lattice contacts unique to the pH 5.4 structure. In addition, the transition from aquomet alpha subunits to bishistidine alpha subunits is accompanied by an approximately 1.2 A movement of the alpha heme groups to a more solvent-exposed position as well as the creation of a solvent channel from the interior of the alpha heme pocket to the outside of the tetramer. These changes and the extensive rearrangement of the crystal lattice structure allow the alpha heme group of one tetramer to make direct contact with an alpha heme group on an adjacent tetramer. These results suggest possible functional roles for hemichrome formation in vivo.     

Influence of globin structures on the state of the heme. Ferrous low spin derivatives.

Studies of high spin ferrous and ferric derivatives led us to conclude that in the quaternary R structure the state of the hemes is similar to that in the free alpha and beta subunits, but in the T structure a tension acts on the hemes which tries to pull the iron and the proximal histidine further from the plane of the porphyrin. We have now studied the effect of inositol hexaphosphate (IHP) on the three low spin ferrous compounds of hemoglobin with O2, CO, and NO. IHP failed to switch the quaternary structure of carbonmonoxy- and oxyhemoglobin A to the T state, but merely caused a transition to an as yet undefined modification of the R structure. IHP is known to cause a switch to the T structure in hemoglobin Kansas. We have found that this switch induces red shifts of the visible alpha and beta absorption bands and the appearance of a shoulder on the red side of the alpha band; these changes are very weak in carbonmonoxy- and slightly stronger in oxyhemoglobin Kansas. As already noted by previous authors, addition of IHP to nitrosylhemoglobin A induces all the changes in uv absorption and CD spectra, sulfhydryl reactivities, and exchangeable proton resonances normally associated with the R leads to T transition, and is accompanied by large changes in the Soret and visible absorption bands. Experiments with nitrosyl hybrids show that these changes in absorption are caused predominantly by the hemes in the alpha subunits. In the accompanying paper Maxwell and Caughey (J. C. Maxwell and W. S. Caughey (1976), Biochemistry, following paper in this issue) report that the NO in nitrosylhemoglobin without IHP gives a single ir stretching frequency characteristic for six-coordinated nitrosyl hemes; addition of IHP causes the appearance of a second ir band, of intensity equal to that of the first, which is characteristic for five-coordinated nitrosyl hemes. Taken together, these results show that the R leads to T transition causes either a rupture or at least a very dramatic stretching of the bond from the iron to the heme-linked histidine, such that an equilibrium is set up between five- and six-coordinated hemes, biased toward five-coordinated hemes in the alpha and six-coordinated ones in the beta subunits. The reason why IHP can switch nitrosyl-, but not carbonmonoxy- or oxyhemoglobin A, from the R to the T structure is to be found in the weakening of the iron-histidine bond by the unpaired NO electron and by the very short Fe-NO bond length.     

Proton NMR study of methemoglobin and its isolated chains. Effect of the subunit association on the structure of the subunits.

In order to investigate the effect of the alpha beta subunit contacts on the subunit structure of human adult methemoglobin, the hyperfine shifted proton NMR spectra of several high spin complexes (water, cyanate, thiocyanate, formate, fluoride, and nitrite) and low spin complexes (imisazole, azide, and cyanide) of hemoglobin and its isolated subunits were characterized at 220 MHz and 22 degrees C. The spectra of ferric low spin derivatives of the isolated subunits were approximately superimposable on the corresponding hemoglobin spectra. On the other hand, the high spin spectra of the isolated subunits were greatly different from each other. The spectral anomaly in the ferric high spin complexes of the isolated beta subunit were interpreted to indicate other structural change than the hemichrome formation in the beta heme pocket. Difference in the subunit association effect between the high and low spin complexes of the isolated beta subunit was interpreted on the basis of a conformational change of the apoprotein dependent on the spin state of the beta heme iron.     

Properties of chemically modified Ni(II)-Fe(II) hybrid hemoglobins. Ni(II) protoporphyrin IX as a model for a permanent deoxy-heme

Chemical modifications, NES-Cys(beta 93), des-Arg(alpha 141), and both modifications on the same molecule, were made to Ni-Fe hybrid hemoglobins, and their effect on individual subunits was investigated by measuring oxygen equilibrium curves, the Fe(II)-N epsilon (His F8) stretching Raman lines, and light-absorption spectra. The oxygen equilibrium properties indicated that modified Ni-Fe hybrid hemoglobins remain good models for the corresponding deoxy ferrous hemoglobins, although K1, the dissociation equilibrium constant for the first oxygen to bind to hemoglobin, was decreased by the chemical modifications. Resonance Raman spectra of deoxy alpha 2 (Fe) beta 2 (Ni) and light-absorption spectra of deoxy alpha 2 (Ni) beta 2 (Fe), revealed that the state of alpha hemes in both hybrid hemoglobins underwent a transition from a deoxy-like state to an oxy-like state caused by these chemical modifications when K1 was about 3 mm Hg (1 mm Hg approximately 133.3 Pa). On the other hand, the state of beta hemes in hybrid hemoglobins was little affected, when K1 was larger than 1 mm Hg. Modified alpha 2 (Fe) beta 2 (Ni) gave a Hill coefficient greater than unity with a maximum of 1.4 when K1 was about 4 mm Hg. The two-state model predicts that the K1 value at the maximum Hill coefficient should be much larger than this value. For oxygen binding to unmodified alpha 2 (Ni) beta 2 (Fe), oxygen equilibrium data suggested no structural change, while the spectral data showed a structural change around Ni(II) protoporphyrin IX in the alpha subunits. A similar situation was encountered with modified alpha 2 (Ni) beta 2 (Fe), although K1 was decreased as a result of the structural changes induced by the modifications.     

Use of heme spin-labeling to probe heme environments of alpha and beta chains of hemoglobin.

A spin label attached to a propionic acid group of the heme has been used to probe the heme environment of the alpha and beta chains of hemoglobin in both the subunit and tetrameric forms. The electron paramagnetic resonance (EPR) studies of hemoglobin hybrids in which the spin label is attached to either the alpha- or beta-heme (alpha2SLbeta 2 or alpha2beta2SL) and spin-labeled isolated chains (alphaSL and betaSL) show that: 1) alpha- and beta-hemes have different environments in the tetrameric forms of oxy-, deoxy-, and methemoglobins as well as in isolated single chains; 2) when isolated subunits associate to form hemoglobin tetramers, the environment of the alpha-heme changes more drastically than that of the beta-heme; 3) upon deoxygenation of hemoglobin, the structure in the vicinity of the alpha-heme changes more drastically than that of the beta-heme; and 4) upon the addition of organic phosphates to methemoglobin, the change in the spin state of the heme irons mainly arises from beta-heme. The results demonstrate conclusively that the alpha and the beta subunits of hemoglobin are structurally nonequivalent as are their structural changes as the result of ligation. The relationship of EPR spectrum and structure of hemoglobin is discussed.     

The stability of the heme-globin linkage in some normal, mutant, and chemically modified hemoglobins.

The rates and equilibria of heme exchange between methemoglobin and serum albumin were measured using a simple new spectrophotometric method. It is based on the large difference between the spectrum of methemoglobin and that of methemealbumin at pH 8-9. The rate of heme exchange was found to be independent of the albumin concentration and inversely proportional to the hemoglobin (Hb) concentration. Taken together with the finding that the rate was 10 times greater for Hb Rothschild, which is completely dissociated into alpha beta dimers and 10 times smaller for two cross-linked hemoglobins, the subunits of which cannot dissociate, this showed that the rate of dissociation of heme from alpha beta dimers is very much greater than from tetramers. Conditions were found for the attainment of an equilibrium distribution of hemes between beta globin and albumin. The equilibrium distribution ratio, R = methemealbumin/albumin/methemoglobin/apohemoglobin, for hemoglobin A was 3.4 with human and 0.005 with bovine serum albumin. Both the rates of exchange and the R values of HbS and HbF were the same as that for HbA. The equilibrium distribution ratio for Hb Rothschild was 7 times greater than that for HbA whereas that of one but not the other of the cross-linked hemoglobins was 10 times smaller. The structural bases for these differences are analyzed.     

Comparative aspects of methemoglobin formation and reduction in opossum (Didelphis virginiana) and human erythrocytes

Glucose-depleted, nitrite-treated opossum erythrocytes effectively reduce methemoglobin in an environment of physiological saline and added glucose does not accelerate the rate of reduction. In autologous plasma or 25 mM phosphate-buffered saline pH 7.4, added glucose significantly accelerates methemoglobin reduction in glucose-depleted, nitrite-treated opossum erythrocytes. Human red cells require added glucose to carry out reduction of methemoglobin and increased phosphate concentration or autologous plasma does not alter the rate of this process. Within the opossum red cell in vitro, autooxidation of hemoglobin proceeds at a much slower rate than that observed in human erythrocytes.     

Differential effects of pH and inositol hexaphosphate on the spectroscopic properties of the alpha and beta subunits in methemoglobins M Milwaukee and A.

The effect of pH and inositol hexaphosphate on the electron spin resonance spectra of the alpha-hemes (g = 6.0) and the beta-hemes (g = 6.7) has been measured in methemoglobin M Milwaukee and compared with that of methemoglobin A (g = 6.0). The beta-hemes are found to be comparatively insensitive to both effectors while the alpha-hemes behave in a manner similar to the heme groups of methemoglobin A. Binding of inositol hexaphosphate enhances the high spin ESR signal of the alpha-hemes in both methemoglobins. Comparison of the optical properties of methemoglobins A and M Milwaukee over the pH range from 5.0 to 8.1 shows that inositol hexaphosphate has a differential effect on the subunit types in these two methemoglobins. At low pH the spectral changes observed upon inositol hexaphosphate binding arise primarily from the beta-hemes, while at neutral and alkaline pH these changes arise from both subunit types. The beta-heme spectral changes appear to be pH independent while those arising from the alpha-hemes are strongly pH dependent. It is concluded that it is the hydroxymet form of the alpha-hemes which undergoes spectral change upon inositol hexaphosphate binding to the beta-subunits. In methemoglobin A the spin state and paramagnetic susceptibility increase only in the neutral and alkaline pH ranges upon inositol hexaphosphate binding (Gupta, R.K. and Mildvan, R.S. (1975) J. Biol. Chem. 250, 246; Perutz, M.F., Sanders, J.K.M., Chenery, D.H., Noble, R.W., Penelly, R.R., Fung, L.W.-M., Ho, C., Giannini, I., Porschke, D. and Winkler, H. (1978) Biochemistry 17, 3640). Therefore the hydroxymet form of the alpha-hemes which is responsible for the observed spectral changes must also be responsible for these increases in the magnetic properties of methemoglobin A. Inositol hexaphosphate can bind to methemoglobin at alkaline pH if the beta-hemes are in the high spin form.     

Spin label studies on conformational changes of aphohemoglobin due to heme binding.

Human apohemoglobin (globin) was spin-labeled at the beta-93 sulfhydryl groups with 2,2,5,5-tetramethyl-3-aminopyrrolidine-I-oxyl. Spin-labeled globin exhibited an EPR spectra that is less immobilized than that of spin-labeled hemoglobin, indicating the conformational difference in the vicinity of the label between hemoglobin and globin. Spectrophotometric titration of spin-labeled globin with protohemin showed that 1 mol of globin (on the tetramer basis) combines with 4 mol of hemin, producing a holomethemoglobin spectrophotometrically indistinguishable from native methemoglobin. The EPR spectrum was also changed strikingly upon the addition of protohemin. This change, however, was not proportional to the amount of hemin added, but marked changes occurred after 3 to 4 mol of hemin were mixed with 1 mol of spin-labeled globin. The EPR spectrum of spin-labeled hemoglobin thus prepared was identical with that prepared by direct spin labeling to methemoglobin. These results suggest the preferential binding of hemin to alpha-globin chains in the course of heme binding by globin. This assumption was further confirmed by preparing spin-labeled semihemoglobin in which only one kind of chain contained hemin (alpha h betaO SL and alpha O beta h SL). The EPR spectrum of the alpha h beta O SL molecule showed a slightly immobilized EPR spectrum, similar to that of spin-labeled globin mixed with 50% of the stoichiometric amount of hemin. On the other hand, the alpha O beta h SL molecule showed a distinctly different EPR signal from that of globin half-saturated with hemin, and showed an intermediate spectrum between those of beta h SL and alpha h beta h SL. These results indicate that heme binding to globin chains brings about a major conformational change in the protein moiety and that chain-chain association plays a secondary role. We conclude that hemin binds preferentially to alpha-globin chains and that the conformation of globin changes rapidly to that of methemoglobin after all four hemes are attached to globin heme pockets.     

Oxygen binding to partially oxidized hemoglobin. Analysis in terms of an allosteric model

We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.     

Conformation and spin state in methemoglobin.

The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: oxygen equilibrium curves and resonance Raman spectra

The structure and function of iron(II)-ruthenium(II) hybrid hemoglobins alpha(Ru-CO)2 beta(Fe)2 and alpha(Fe)2 beta(Ru-CO)2, which can serve as models for the intermediate species of the oxygenation step in native human adult hemoglobin, were investigated by measuring oxygen equilibrium curves and the Fe(II)-N epsilon (His F8) stretching resonance Raman lines. The oxygen equilibrium properties indicated that these iron-ruthenium hybrid hemoglobins are good models for the half-liganded hemoglobin. The pH dependence of the oxygen binding properties and the resonance Raman line revealed that the quaternary and tertiary structural transition was induced by pH changes. When the pH was lowered, both the iron-ruthenium hybrid hemoglobins exhibited relatively higher cooperativity and a Raman line typical of normal deoxy structure, suggesting that their structure is stabilized at a "T-like" state. However, the oxygen affinity of alpha(Fe)2 beta(Ru-CO)2 was lower than that of alpha(Ru-CO)2 beta(Fe)2, and the transition to the "deoxy-type" Fe-N epsilon stretching Raman line of alpha(Fe2)beta(Ru-CO)2 was completed at pH 7.4, while that of the complementary counterpart still remained in an "oxy-like" state under the same condition. These observations clearly indicate that the beta-liganded hybrid has more "T"-state character than the alpha-liganded hybrid. In other words, the ligation to the alpha subunit induces more pronounced changes in the structure and function in Hb than the ligation to the beta subunit. This feature agrees with our previous observations by NMR and sulfhydryl reactivity experiments. The present results are discussed in relation to the molecular mechanism of the cooperative stepwise oxygenation in native human adult hemoglobin.     

Intrinsic fluorescence of carp hemoglobin: a study of the R----T transition

The intrinsic fluorescence of hemoglobins is known to respond to ligand-induced changes in the quaternary structure of the protein. Carp hemoglobin is an interesting model to study the quaternary transition since its R----T equilibrium is pH-dependent and at low pH, in the presence of organic phosphate, it remains in the T or 'deoxy' quaternary structure, even when saturated with ligand. In this study, using front-face fluorometry, we show that the intrinsic fluorescence intensity exhibited by carp carboxyhemoglobin increases as the pH is lowered below 6.5 in the presence of inositol hexaphosphate. At low pH, carp methemoglobin is less affected by the addition of inositol hexaphosphate than is the CO derivative, while little or no change is observed in the met-azide derivative. We conclude: (1) the exact nature of the R to T state transition induced by inositol hexaphosphate differs for carp carboxy-, met- and met-azide hemoglobin derivatives; (2) the chromophores responsible for the changes observed with absorption spectroscopy may not be the same as those chromophores responsible for the fluorescence differences; and (3) alpha 46-Trp is tentatively assigned as one source of fluorescence emission. Furthermore, fluorescence properties of carp hemoglobin are compared to those of human hemoglobin.     

Electron transfer kinetics between hemoglobin subunits

The kinetics for electron transfer have been measured for samples of hemoglobin valency hybrids with initially one type of subunit, alpha or beta, in the oxidized state. Incubation of these samples under anaerobic conditions tends to randomize the type of subunit that is oxidized. With a time coefficient of a few hours at pH 7, 25 degrees C, the Hb solution (0.1 mm heme) approaches a form with about 60% of beta chains reduced, indicating a faster transfer rate in the direction alpha to beta. There was no observable electron transfer for samples saturated with oxygen. The electron transfer occurs predominantly between deoxy and aquo-met subunits, both high spin species. Furthermore, electron transfer does not depend on the quaternary state of hemoglobin. Incubation of oxidized cross-linked tetramer Hb A with deoxy Hb S also displayed electron transfer, implying a mechanism via inter-tetramer collisions. A dependence on the overall Hb concentration confirms this mechanism, although a small contribution of transfer between subunits of the same tetramer cannot be ruled out. These results suggest that in vivo collisions between the Hb tetramers will be involved in the relative distribution of the methemoglobin between subunits in association with the reductase system present in the erythrocyte.     

NMR studies of the quaternary structure and heterogeneity of nitrosyl- and methemoglobin.

NMR was used to study the quaternary structure of nitrosyl- and methemoglobin, the kinetics and equilibrium behavior of nitric oxide binding, and the oxidation of hemoglobin. The -9.6 ppm (from H2O) resonance was used as a measure of nitrosylhemoglobin molecules in the T quaternary structure. We found that stripped nitrosylhemoglobin is 70% in the T state below pH 6.4, and is in the R state above. Inositol hexaphosphate (IHP) raises this transition point to pH 7.5. For stripped aquomethemoglobin, the T marker at -10 ppm is absent. In IHP, at pH 6.5 all of the molecules are in the T state. At both higher and lower pH they shift to the R state. The intensity decreases to half of its maximum at pH 5.5 and 7.4. The relative affinity of nitric oxide binding to the alpha and beta subunits was inferred from the intensities of the resonances at -12 and -18 ppm. Under conditions in which nitrosylhemoglobin exists in the T state, NO binds to the alpha subunit 10 times more strongly than it does to the beta subunit. The kinetic experiments reveal that it binds to the two subunits at the same rate and that it dissociates at 5 x 10(-3) s-1 from the beta subunit and at 5 x 10(-4) s-1 from alpha subunit. At high pH, the two subunits are ligated at the same rate. Potassium ferricyanide oxidation, at pH 6.0 in the absence of IHP, is about 3 times more favorable for the alpha than the beta subunit. Addition of IHP raises this preferential oxidation slightly. At pH 8.44, both alpha and beta subunits were oxidized at the same rate.     

Tertiary structure variability within the quaternary states of hemoglobin: a spin label study

Using variable temperature techniques, the spin label spectral resolution of hemoglobin labeled at the beta93 cysteines with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodonacetamide has been greatly enhanced. The effects of different ligands, inositol hexaphosphate, pH and salt concentration upon spin labeled ferrous and ferric hemoglobin indicate that the beta chain tertiary structure exhibits considerable variability within the oxy and deoxy quaternary structures. From these studies ligand and spin state changes both appear to be of significance in producing structural changes; binding of inositol hexaphosphate then produces further structural changes secondary in amplitude.     

Assessment of the alpha 1 beta 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (alpha 2+ beta 2 and alpha 2 beta 2+: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the alpha 1 beta 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both alpha 2+ beta 2 and alpha 2 beta 2+ showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the alpha 1 beta 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.     

A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.