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.     

Proton nuclear magnetic resonance investigation of the allosteric transition in ligated and unligated carp hemoglobin. Evidence for structural heterogeneity in the heme pocket

The proton nuclear magnetic resonance spectra of carp hemoglobin (Hb) in the unligated deoxy and ligated met-cyano and met-azido forms have been recorded as a function of pH and upon addition of inositol hexaphosphate. All protein derivatives yield spectra that are consistent with appreciable molecular heterogeneity in the heme cavity. The pattern of heme methyl hyperfine shifts in carp met-cyano Hb indicates that this heterogeneity arises from the presence of heme rotational disorder, as found in native myoglobin. In carp deoxy Hb, the T----R transition manifests itself in nuclear magnetic resonance spectral changes similar to those found in modified human Hb species; namely, a decrease in heme methyl and an increase in proximal histidyl imidazole ring NH hyperfine shifts indicative of a strengthening of the iron-histidine bond. The met-cyano complex exhibits heme methyl hyperfine shifts similar to the analogous R state complex of Hb A; addition of inositol hexaphosphate did not give evidence for a quaternary structural change. Carp met-azido Hb in the R state also closely resembles the electronic structure of the HbA complex. Addition of inositol hexaphosphate appeared to effect at least a partial conversion to a T state with larger high-spin content than that observed for T state human metHbN3.     

Effects of ligand size on pH and organic phosphate-dependent affinity changes in carp hemoglobin as measured by isonitrile binding

The equilibria of the binding of methyl and ethyl isonitrile to carp hemoglobin have been measured at three pH values in the presence and absence of inositol hexaphosphate. The binding of methyl isonitrile is characterized by a higher overall dissociation constant, C1/2, and a higher Hill coefficient, n, than that of the ethyl derivative. The former is consistent with the greater hydrophobicity of ethyl isonitrile, and the latter is probably due to a greater intrinsic difference or heterogeneity in the binding affinities of the alpha- and beta-chains for the larger ligand. Changes in log C1/2 which result from alterations in pH or addition of organic phosphate are the same for both ligands within experimental error. This result is not consistent with affinity changes being the result of steric interactions between the protein and the ligand. At pH 6 in the presence of inositol hexaphosphate, equilibrium parameters estimated from overall rates of ligand binding and dissociation are in good agreement with direct equilibrium measurements. This is consistent with the protein being in a low-affinity, T-like state even when saturated with ligand under these conditions, resulting in a loss of cooperativity in ligand binding. At high pH, ligand binding remains cooperative, as evidenced by n values greater than unity, a general lack of agreement between measured equilibrium parameters and those estimated from overall kinetic constants, and differences in the kinetics of ligand binding as observed by rapid-mixing and flash photolysis techniques. Thus, the deoxygenated state of carp hemoglobin at high pH does not appear to be a good model of a deoxygenated R quaternary structural state.     

Ligand binding kinetic studies on the hybrid hemoglobin alpha (human):beta (carp): a hemoglobin with mixed conformations and sequential conformational changes

Oxygen and CO ligand binding kinetics have been studied for the hybrid hemoglobin (Hb) alpha (human):beta (carp), hybrid II. Valency and half-saturated hybrids were used to aid in the assignment of the conformations of both chains. In hybrid II, an intermediate S state occurs, in which one chain has R- and the other T-state properties. In HbCO at pH 6 (plus 1 mM inositol hexaphosphate), the human alpha-chain is R state and the carp beta-chain is T state. We have no evidence at this pH that the carp beta-chain ever assumes the R conformation. At pH 6, the human alpha-chain shows human Hb R-state kinetics at low fractional photolysis and T-state rates for CO ligation by stopped flow. At pH 7, the human-chain R-state rate slows toward a carp hemoglobin rate. The carp beta-chains, on the other hand, react 50% more rapidly in the liganded conformation than in carp hemoglobin, and while the human alpha-chains are in the R state, the two beta-chains appear to function as a cooperative dimer. In this hemoglobin, the chains appear to be somewhat decoupled near pH 7, allowing a sequential conformational change from the R state in which the beta-chains first assume T-state properties, followed by the alpha-chains. The rate of the R-T conformational change for the carp beta-chains is at least 300 times greater than that for the human alpha-chains. At pH 9, the R----T conformational transition rate is at least 200 times slower than that for human hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural states and transitions of carp hemoglobin.

The wide ligand affinity range previously observed for carp hemoglobin is bounded at both extremes by regions of constant affinity. Within these regions, pH, organic phosphates, and the extent of ligand binding have no effect on the measured affinity and the cooperativity of ligand binding is greatly reduced or absent. The rates of CO recombination to fully and partially unliganded carp hemoglobin, under various organic phosphate and pH conditions, are shown to reflect this behavior. Constant kinetic rates are seen to directly correspond to the regions of constant affinity. Therefore, these are taken to be single protein conformations, one of high and one of low ligand affinity. In the simplest view, these conformations represent the R and T states of a two-state model, and most of the properties of carp hemoglobin are explained quite well within this framework. Increases in either hydrogen or phosphate ion concentrations favor the stabilization of the low affinity structure of even fully liganded carp hemoglobin. We have studied the structural transition from high to low affinity by monitoring the absorption spectra of carp hemoglobins at constant pH as a function of organic phosphate concentration. We find that different spectra are induced in both carp methemoglobin and cyanomethemoglobin by inositol hexaphosphate addition. Furthermore, the dependence of the magnitude of the spectral changes on pH and organic phosphate concentration is the close agreement with that predicted from studies of the ligand binding properties of the molecule.     

Gelation of sickle hemoglobin. III. Nitrosyl hemoglobin.

Sickle cell nitrosyl hemoglobin was examined for gelation by an ultracentrifugal method previously described (Briehl &; Ewert, 1973) and by birefringence. In the presence of inositol hexaphosphate gelation which exhibited the endothermic temperature dependence seen in gels of deoxyhemoglobin S was observed by both techniques. In the absence of inositol hexaphosphate no gelation was observed, nor did nitrosyl hemoglobin A exhibit gelation. On the assumption that gelation is dependent on the deoxy or T (low ligand affinity) as opposed to the oxy or R (high ligand affinity) quaternary structure this supports the conclusion that nitrosyl hemoglobin S in inositol hexaphosphate assumes the T structure, in contrast to the other liganded ferrohemoglobin derivatives oxy and carbon monoxide hemoglobin. Assuming further that the quaternary structures and isomerizations are the same in hemoglobins A and S it can also be concluded that nitrosyl hemoglobin A in inositol hexaphosphate assumes the T state. Since no gelation was seen in stripped nitrosyl hemoglobin S, inositol hexaphosphate serves to effect an R to T switch in this derivative. Thus R-T isomerization in nitrosyl hemoglobin occurs without change in ligand binding at the sixth position of the heme group confirming the conclusion of Salhany (1974) and Salhany et al. (1974).Lowering of the pH toward 6 favors gelation of NO hemoglobin S as it does of deoxy and aquomethemoglobin S (Briehl &; Ewert, 1973,1974), consistent with a favoring of the T structure due to strengthening of the interchain salt bridges and the binding of inositol hexaphosphate and/or changes in site-to-site interactions on which gelation depends.     

Stabilization of the T-state of hemoglobin

The effect of inositol hexaphosphate and bezafibrate on binding of O2 and CO to HbAO at high concentrations (1 mM) has been evaluated using thin layer optical techniques. Data analysis shows 1) the occurrence of greatly reduced ligand dependent cooperativity (Hill slope of 2.23 for CO and 1.51 for O2), and 2) the presence of significant triply ligated species. The data fits a nested allosteric two-state MWC model in which the T state consists of two allosteric substrates, Tt and Tr, where Tt binds only to the alpha chains and Tr binds to both alpha and beta chains. The model indicates that the triply ligated species consists of a predominant amount of T form, agreeing with kinetic observations of CO ligated hemoglobin. The maximum amount of triply ligated R molecules (CO or O2) implicated is less than 1%, a result similar to that found previously for binding studies made in the absence of BZF and IHP.     

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.     

The effects of inositol hexaphosphate on the allosteric properties of two beta-99-substituted abnormal hemoglobins, hemoglobin Yakima and hemoglobin Kempsey.

Hemoglobins (Hb) Yakima and Kempsey were purified from patients' blood with diethylaminoethyl cellulose column chromatography. The oxygen equilibrium curves of the two hemoglobins and the effects of organic phosphates on the function were investigated. In 0.1 M phosphate buffer, Hill's constants n for Hb Yakima and Hb Kempsey were 1.0 to 1.1 at the pH range for 6.5 to 8.0 and the oxygen affinities of both the mutant hemoglobins were about 15 to 20 times that of Hb A at pH 7.0. The Bohr effect was normal in Hb Yakima and one-fourth normal in Hb Kempsey. In the presence of inositol hexaphosphate, the oxygen affinities to Hb Yakima and Hb Kempsey were greatly decreased, and an interesting result revealed that these hemoglobins showed clear cooperativity in oxygen binding. Hill's constant n in the presence of inositol hexaphosphate was 1.9 for Hb Kempsey and 2.3 for Hb Yakima at pH 7.0. The cooperativities of these mutant hemoglobins were pH-dependent, and Hb Kempsey showed high cooperativity at low pH (n equal 2.1 at pH 6.6) and low cooperativity at high pH (n equal 1.0 at pH 8.0). Hb Yakima showed similar pH dependence in cooperativity. In the presence of inositol hexaphosphate, Hb A showed a pH-dependent cooperativity different from those of Hb Yakima and Hb Kempsey, namely, Hill's n was the highest in alkaline pH (n equal 3.0 at pH 8.0) and decreased at lower pH (n equal 1.5 at pH 6.5). 2,3Diphosphoglycerate bound with the deoxygenated Hb Yakima and Hb Kempsey, however, had no effect on the oxygen binding of these abnormal hemoglobin. The pH-dependent cooperativity of alpha1beta2 contact anomalous hemoglobin and normal hemoglobin was explained by the shifts in the equilibrium between the high and low ligand affinity forms.     

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.     

Recombinant hemoglobins with low oxygen affinity and high cooperativity

By introducing an additional H-bond in the alpha(1)beta(2) subunit interface or altering the charge properties of the amino acid residues in the alpha(1)beta(1) subunit interface of the hemoglobin molecule, we have designed and expressed recombinant hemoglobins (rHbs) with low oxygen affinity and high cooperativity. Oxygen-binding measurements of these rHbs under various experimental conditions show interesting properties in response to pH (Bohr effect) and allosteric effectors. Proton nuclear magnetic resonance studies show that these rHbs can switch from the oxy (or CO) quaternary structure (R) to the deoxy quaternary structure (T) without changing their ligation states upon addition of an allosteric effector, inositol hexaphosphate, and/or reduction of the ambient temperature. These results indicate that if we can provide extra stability to the T state of the hemoglobin molecule without perturbing its R state, we can produce hemoglobins with low oxygen affinity and high cooperativity. Some of these rHbs are also quite stable against autoxidation compared to many of the known abnormal hemoglobins with altered oxygen affinity and cooperativity. These results have provided new insights into the structure-function relationship in hemoglobin.     

Circular dichroism as a probe of the allosteric R in equilibrium T transformation in hemoglobins and modified hemoglobins.

The circular dichroism spectra at pH 6.5 of a number of hemoglobins and modified hemoglobins have been recorded in the 280 nm region and interpreted in terms of shifts of the R?T allosteric transformation. Inositol hexaphosphate converts aquomet hemoglobin A(S) to the T form but the carbamlyated derivatives are unaffected by inositol hexaphosphate and remain in the R form. Fluorodinitrobenzene and dimethyl adipimidate modified hemoglobins are locked in an intermediate form, and inositol hexaphosphate has little or no effect. The circular dichroism in the 280 nm region is shown to be a useful diagnostic tool for chemical agents that affect the R?T allosteric transformation.     

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin.

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.     

Quaternary structure and geminate recombination in hemoglobin: flow-flash studies on alpha 2CO beta 2 and alpha 2 beta 2CO.

The kinetics of geminate recombination for the diliganded species alpha 2CO beta 2 and alpha 2 beta 2CO of human hemoglobin were studied using flash photolysis. The unstable diliganded species were generated just before photolysis by chemical reduction in a continuous flow reactor from the more stable valency hybrids alpha 2CO beta 2+ and alpha 2+ beta 2CO, which could be prepared by high pressure liquid chromatography. Before the flash photolysis studies, the hybrids had been characterized by double-mixing stopped-flow kinetics experiments. At pH 6.0 in the presence of inositol hexaphosphate (IHP) both of the diliganded species show second order kinetics for overall addition of a third CO that is clearly characteristic of the T state (l' = 1-2 x 10(5) M-1 s-1), whereas at higher pH and in the absence of IHP they show combination rates characteristic of an R state. The kinetics of geminate recombination following photolysis of a bound CO, however, showed little dependence on pH and IHP concentration. This surprising observation is explained on the basis that the kinetics of geminate recombination of CO primarily depends on the tertiary structure of the ligand binding site, which apparently does not differ much between the R state and the liganded T state formed on adding IHP in this system. Since this explanation requires distinguishing different tertiary structures within a particular quaternary structure, it amounts to a contradiction to the two-state allosteric model.     

Mutations of the betaN102 residue of HbA not only inhibit the ligand-linked T to Re state transition, but also profoundly affect the properties of the T state itself

The properties of three HbA variants with different mutations at the beta102 position, betaN102Q, betaN102T, and betaN102A, have been examined. All three are inhibited in their ligand-linked transition from the low affinity T quaternary state to the high affinity Re quaternary state. In the presence of inositol hexaphosphate, IHP, none of them exhibits cooperativity in the binding of oxygen. This is consistent with the destabilization of the Re state as a result of the disruption of the hydrogen bond that normally forms between the beta102 asparagine residue and the alpha94 aspartate residue in the Re state. However, these three substitutions also alter the properties of the T state of the hemoglobin tetramer. In the presence of IHP, the first two substitutions result in large increases in the ligand affinities of the beta-subunits within the T state structure. The betaN102A variant, however, greatly reduces the pH dependencies of the affinities of the alpha and beta subunits, K1(alpha) and K1(beta), respectively, for the binding of the first oxygen molecule in the absence of IHP. In the presence of IHP, the T state of this variant is strikingly similar to that of HbA under the same conditions. For both hemoglobins, K1(alpha) and K1(beta) exhibit only small Bohr effects. In the absence of IHP, the affinities of the alpha and beta subunits of HbA for the first oxygen are increased, and both exhibit greatly increased Bohr effects. However, in contrast to the behavior of HbA, the ligand-binding properties of the T state tetramer of the betaN102A variant are little affected by the addition or removal of IHP. It appears that along with its effect on the stability of the liganded Re state, this mutation has an effect on the T state that mimics the effect of adding IHP to HbA. It inhibits the set of conformational changes, which are coupled to the K1 Bohr effects and normally accompany the binding of the first ligand to the HbA tetramer in the absence of organic phosphates.     

Chain equivalence in reaction of nitric oxide with hemoglobin.

Mixtures of nitric oxide and hemoglobin were prepared in a rapid freeze apparatus and analyzed by EPR spectroscopy. Spectra from samples at various degrees of saturation showed that the two subunits bound NO at equal rates. Identical results were observed in 0.1 M phosphate at pH 6.5 and 0.1 M 2,2'-bis(hydroxymethyl)-2,2',2'-nitrilotriethanol, 0.1 M NaCl at pH 7.0, both in the presence and absence of inositol hexaphosphate at either buffer condition. At subsaturating levels of NO (less than 60%), or at all levels of saturation in the presence of inositol hexaphosphate, it was found that the EPR spectrum of nitrosylhemoglobin varied with the length of time before freezing. This change was characterized by the development of a hyperfine structure at g = 2.01 which appeared with a half-time of approximately 0.4 s. Maxwell and Caughey (Maxwell, J. C., and Caughey, W. S. (1976) Biochemistry 15, 388-395) have attributed this three-line EPR hyperfine structure to the formation of a pentacoordinate ferroheme-NO complex. Corresponding slow changes were observed in the visible absorption spectrum following the binding of low levels of NO to deoxyhemoglobin or inositol hexaphosphate to fully saturated nitrosylhemoglobin. Thus it appears that NO binding to the alpha and beta subunits of deoxyhemoglobin takes place at equal rates and, under conditions favoring the T quaternary state (low saturation, presence of inositol hexaphosphate), a further slow structural change takes place, resulting in the cleavage of the iron--proximal histidine bond.     

High and low oxygen affinity conformations of T state hemoglobin

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15 degrees C, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.     

Nuclear relaxation studies on human methemoglobin. Observation of cooperativity and alkaline Bohr effect with inositol hexaphosphate.

Ehanced spin-lattice relaxation (1/t1) of water protons induced by the heme iron of human aquomethemoglobin is exchanged-limited (koff = 1.4 times 10-4 per s at 30 degrees, H+ =7.5 Cal per mol) as indicated by the temperature and frequencey dependencies. A comparison of deuteron and proton relaxation rates revealed an order of magnitude primary isotope effect and a small inverse secondary isotope effect on the escape rate of protons from the heme iron into bulk water establishing the exchange of protons and not the exchange of the entire water molecule to be the chemical mechanism of the entire water molecule to be the chemical mechanism of the exchange process. With fluoromethemoglobin, the relaxation rate is in the fast exchange region. The results can be understood in terms of a water molecule interacting with the heme iron at an iron to proton distance less than 3.4 A in aquomethemoglobin and a single proton at a distance of 4.11 A assignable to the NH proton of the distal histidine imidazole group in fluoromethemoglobin. The relaxation rates are pH-dependent and normal titrations with Hill coefficients n = 1 are observed. The pKa is less than or equal to 6. 7 with aquomethemoglobin and 8.5 with fluoromethemoglobin at 30 degrees C. The binding of inositol hexaphosphate in stoichiometric amounts has no significant effect on the magnetic susceptibility of solutions of aquomethemoglobin and fluoromethemoglobin, but in the former case it increases koff to 3.8 times 10-4 per s by lowering the H+ barrier to 6.8 Cal per mol. In fluoromethemoglobin, inositol hexaphosphate decreases the iron to distal histidine NH distance by 0.17 A and the electron relaxation time taus by 10% as determined by the frequency dependence of 1/T1. In the aquomethemoglobin system, inositol hexaphosphate induces a Bohr effect, raising the pKa of the ionization responsible for the 1/T1 titration to 7.2, and induces cooperativity in the pH titration with a Hill coeffocoemt n = 2.8 plus or minus 0.1. With fluoromethemoglobin, the normal pH titration curve is unaffected by inositol hexaphosphate (n approximately equal to 1). Further, relaxivity titrations with varying amounts of azide and fluoride near neutral pH show normal behavior (n = 1) with and without inositol hexaphosphate. These results indicated that inositol hexaphosphate alters the quaternary structure of methemoglobin to the deoxy conformation without causing a change in the spin state of the heme iron...     

The relative electron affinities of the alpha and beta chains of oxyhaemoglobin as a function of pH and added inositol hexaphosphate. An electron spin resonance study.

Exposure of aqueous glasses of oxyhaemoglobin to 60Co gamma-rays at 77K results in electron addition to the FeO2 unit, the ESR spectrum for the alpha-chain electron adduct being well separated from that for the beta-chain. The relative yields of these two centres has been measured in the pH range 4.5 to 8.5, with or without added inositol hexaphosphate. We find that, in the absence of inositol hexaphosphate, the yield of beta-chain adduct is almost equal to that of the alpha-chains in the pH 4--5 region, but these rapidly diverge with increasing pH, the beta-yield increasing and the alpha-yield decreasing. After a plateau in the pH 6--8 region, the yield of beta-chain adduct decreases, but that of the alpha-chain adduct remains constant. In the presence of an excess of inositol hexaphosphate the pH change for the beta-adduct remains, but at low pH values the yield of the alpha-adduct is much greater than that of the beta-adduct. This constraint is removed with a pK of approx. 7.7 and at high pH values the yield of the beta-adduct is once again greater than that of the alpha-adduct. These results are significant in that they suggest that the electron affinities of the alpha and beta chains in oxyhaemoglobin are a function of pH, with that of the beta-chains being greater than that of the alpha-chains in the neutral region. Also inositol hexaphosphate clearly binds to one or both chains, and this has the effect of reversing the relative electron affinities of the two chains.