Absence Of IHP Research Articles

Functional Properties of Hb S and Hb C in Stored Cpda-1 Red Blood Cells Concentrate — a Prospective Study

Most of the 1,300 human hemoglobin (Hb) variants are characterized by normal O2 binding, including here the highly prevalent Hbs S [HBB:c.20A>T; p.Glu6Val] and C [HBB:c.19G>A; p.Glu6Lys]. Hb S and Hb C carriers are generally asymptomatic and might be admitted as blood donors. However, the functional efficiency of these variants in standard blood bags - red blood cells (RBC) concentrates - under the interference of anticoagulant and storage is not well established. In this prospective study, three samples of RBC concentrates (phenotypes AA, AS and AC), with citrate phosphate dextrose adenine (CPDA-1) as anticoagulant, were analyzed for equilibrium tests and compared to fresh Hb A without anticoagulant. The Hb samples were extracted after 48 hours and 35 days of storage and purified by exclusion and ion exchange chromatography (Sephadex G-25 and Amberlite MB-3 columns in Hepes buffer without NaCl). O2 affinity was evaluated by spectrophotometry-tonometry method, by determining the p50 in stripped lysates ([Hb]=70 μM/Heme), in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) in all studied pHs. The activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. The p50 of Hb A after 35 days in stored blood bag was decreased in all pHs, in both presence and absence of IHP, when compared to fresh Hb A, suggesting that the anticoagulant and the storage might affect HbA-O2 binding, increasing its affinity for the ligand. No other differences were observed: heme-heme cooperativity and Bohr effect were similar to fresh Hb A without anticoagulant. The same was found when stored Hb AS was compared to Hb A from blood bag (35 days storage, in the stripped state and under the interference of IHP). However, the stripped lysate containing Hb AC revealed an important reduction of the O2 affinity after both 48 hours and 35 days of storage, mainly at pHs ³ 7, thus interfering in the Bohr effect. The heme-heme cooperativity resulted in normal n coefficients, indicating that the Hb-O2 ligation remained cooperative, in spite of the significant reduction of O2 affinity. Similar results were observed in the presence of IHP (48 hours and 35 days), although Bohr effect was reestablished, indicating that the supplementation with a 2,3-BPG-like allosteric effector might restore some Hb-ion interactions, and therefore, Hb C function. As the 2,3-BPG is consumed with storage of RBC concentrates, Hb AC showed an even lower affinity for O2 after 35 days of storage. Although still preliminary, our results indicate a significant functional reduction of Hb C in RBC concentrates in standard blood bag (CPDA-1 anticoagulant), proportional to the time of storage. Considering that the screening for hemoglobinopathies in the blood banks routine is mainly focused in the detection of Hb S, a greater understanding about the properties of native Hb and its most frequent variants in stored RBC concentrates could also contribute with the determination of safe parameters in hemotherapy based on the functional efficiency of each hemoglobin. Financial Support: Fapesp, CNPq, Capes, Faepex-Unicamp. DisclosuresNo relevant conflicts of interest to declare.

Mutations of the βN102 Residue of HbA Not Only Inhibit the Ligand-Linked T to ReState 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.

Metal ion coordination in ‘R’ and ‘T’ state hybrid hemoglobins as revealed by optical, EPR and sulphhydryl reactivity studies

The sulphhydryl environment in various mixed-metal hybrid hemoglobins, viz. α2(Cu)-β2(FeCO), α2(FeCO)-β2(Cu), α2(Cu)-β2(Ni), α2(Ni)-β2(Cu), was studied by reacting them with the sulphhydryl reagent, 4,4′-dithiodipyridine (4-PDS). The reactivity was compared with that of HbCO, NiHb and CuHb. It is found that there exists a correlation between conformational change and metal ion environment, not only at the extreme R and T states but also the intermediate conformations. EPR examinations of these hybrids show that both in R state-[Cu(II)-Fe(II)] and T state-[Cu(II)-Ni(II)] hybrids at neutral pH and in the absence of IHP, CuPPIX, irrespective of the subunit in which it is present, has a mixed-metal ion environment: Species 1, a five-coordinated Cu2+ complex with strong proximal histidine bond and species 2, a four-coordinated complex without any covalent linkage with Nε F8-histidine.

Oxygen Equilibrium and EPR Studies on 1 1 Hemoglobin Dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin.

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.

Oxygen equilibrium studies of cross-linked iron-cobalt hybrid hemoglobins. Models for partially ligated intermediates of cobalt hemoglobin.

To probe the molecular mechanism of allosteric function of cobaltous protoporphyrin-substituted hemoglobin (CoHb), a series of alpha, alpha-cross-linked symmetric and asymmetric Fe-Co hybrid hemoglobins, which contain (1Co porphyrin/3Fe porphyrins), (2Co porphyrins/2Fe porphyrins), and (3Co porphyrins/1Fe porphyrin) per tetramer, have been prepared. Because only Fe porphyrin-containing subunits react with CO, these Fe-Co hybrids are converted to mono-, di-, and tri-CO-ligated states in the presence of CO, respectively, and are proposed to stand as models for mono-, di-, and tri-ligated intermediates of CoHb, respectively. The oxygen binding properties of these Fe-Co hybrids were investigated by measuring oxygen binding isotherms in the presence of CO as a function of pH in the presence and absence of IHP. The ligation of CO to a beta subunit causes larger changes in the oxygen affinity and the Bohr effect than that to an alpha subunit, indicating that the ligation to a beta subunit induces larger affinity-related structural changes in cross-linked CoHb. Di- and tri-CO-ligated intermediates exhibited substantially increased oxygen affinity, reduced Bohr effect, and reduced IHP effect, indicating that they are in high affinity states. Calculation of the Adair equilibrium constants for the first and last oxygenation steps for each of these intermediates permitted the determination of the level of free energy of cooperation. The intermediately ligated species of cross-linked CoHb are distributed in multiple levels of free energy of cooperation within the free energy difference of 1.14 kcal mol-1 between deoxy and fully ligated states of cross-linked CoHb at pH 7.4. The ligation process in CoHb is determined by the number and distribution of the bound ligands, and ligation takes place through steps that require minimal free energy changes.

Covalent fixation of polymer-linked benzene hexacarboxylate onto human haemoglobin

The reaction of human deoxy and oxyhaemoglobin with a macromolecular effector, monomethoxypolyoxyethylene-linked benzene hexacarboxylate, in the presence of a water soluble carbodiimide, produces under defined conditions, thesame conjugates preferentially acylated at the two valines β1. The oxygen affinity of both these conjugates is decreased by approximately 5-fold compared with that of native Hb (at pH 7.2, in 0.05 M Tris buffer. 25°C, P 50:20.1 and 20.7 Torr versus about 4 Torr for Hb). This difference appears to be due to an overstabilization of the T state probably together with a decreased of the oxygen affinity of the R state. Addition of the IHP to the conjugate solutions does not influence the P 50 but addition of IHP to the reaction mixtures before the coupling limits the substitution of Hb by the macromolecular effector, to 20% (instead of 100% in absence of IHP). The cooperativity curve is shifted to the right with an n max of 3 at about 90% oxygen saturation, which corresponds to a potential release of 48% of oxygen at pH 7.2,25°C, between 100 and 40 Torr, compared with 40% for blood. Such kinds of conjugates especially those obtained from oxyhaemoglobin which are easily prepared, could be of a great interest as non-diffusing oxygen carriers in transfusional and perfusional fluids.

Enzymatic methemoglobin reduction - effect of organic phosphate

The enzymatic reduction of aquomethemoglobin A, A 1C, fluoro-methemoglobin A (high spin) and cyanomethemoglobin A (low spin) by NADH-methemoglobin reductase was studied in the presence and absence of IHP and NaCl. It is shown that at alkaline pH, IHP accelerates the rate of reduction of high spin methemoglobins only. This effect is specific for IHP and cannot be produced by NaCl, although NaCl does exert similar effect as IHP at acid pH. Blocking of the NH 2- termini of β-chains (Hb A 1C) does not alter the effect of IHP on methemoglobin reduction.

The dissociation of NO from nitrosylhemoglobin.

The reaction between nitrosylhemoglobin and an excess of deoxymyoglobin has been used to study the kinetics of ligand dissociation from Hb4(NO)4 and Hb4(no)1 species. The kinetics of the dissociation of the first NO molecule from Hb4(no)4 was studied by the ligand replacement method. The results indicate that: (a) the ligand dissociation reaction in Hb4(NO)4 is a cooperative process. This is consistent with the results of Moore and Gibson (Moore, E.G., and Gibson, Q.H. (1977) J. Biol. Chem. 251, 2788-2794). (b) alpha and beta chains in the T state formed by adding IHP to Hb4(NO)4 show kinetic heterogeneity. (c) A similar kinetic heterogeneity is shown by alpha and beta chains in the species Hb4NO in the absence of IHP.(d) The value for the NO dissociation rate constant calculated from the slow phases observed in (b) and (c) is similar to that estimated for the R state. These results suggest that the R to T transition brought about with or without inositol hexaphosphate changes the ligand affinity of one type of the chains much more than of the other. On the basis of IR and EPR studies, it is suggested that alpha chains undergo larger functional changes in R to T transition (or vice versa) in nitrosylhemoglobin. The kinetic parameters for HbNO are compared with those of HbO2 and HbCO and the implications of the results for the reaction mechanism are discussed.

Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity: Changes in viscosity and volume on aggregation

The kinetics of gelation of deoxyhemoglobin S were investigated as a function of temperature, concentration of hemoglobin, and solvent composition. Measurements were made by continuously monitoring the changes in viscosity with time, after polymerization had been induced by rapidly raising the temperature. A specially constructed low-shear viscometer was used. The solution density was also measured continuously to determine whether a volume change accompanied aggregation. The results confirm earlier work in showing that the time-dependence of the viscosity is composed of a variable latent period (several minutes to tens of hours) during which there is only a slight and very gradual increase in viscosity, followed by a stage in which the viscosity rises very sharply within a very short time. The length of the initial latent period is highly dependent upon the HbS ‡ ‡ Abbreviations used: Hb, hemoglobin; IHP, inositol hexaphosphate concentration (33rd ± 6 power) and temperature. If the duration is interpreted as the inverse of a reaction rate, the activation energy is 96 ± 10 kcal/mol for solutions containing inosital hexaphosphate. Unlike measurements reported by others, no dependence of the latent period on shear rate was observed at the low shear rate employed. When IHP is omitted from the hemoglobin solutions, qualitatively similar results are obtained; however, the latent period depends on the 26th ± 6 power of the deoxyhemoglobin S concentration and yields an average activation energy of 125 ± 10 kcal/mol. The length of the latent period is increased 40-fold. Tris is known to prevent gelation but the inhibition can be partly reversed by adding IHP. When this is done, highly concentration-dependent latent periods are again observed. The results may be interpreted in terms of nucleation kinetic theories: a critical nucleus composed of approximately 30 hemoglobin molecules is required for gelation; and the energy barrier (which is larger in the absence of IHP) to the formation of this critical aggregate is approximately 100 kcal/mol. Gelation is not accompanied by a detectable volume change (limits 5 × 10 −5 g/ml). This indicates that the volume change of the reaction must be less than + 60 cm 3/mol when the aggregates represent one half of the HbS available for polymerization.

Relations between optical spectrum and structure in nitrosyl hemoglobin and hybrids.

The addition of inositolhexaphosphate to stripped nitrosyl hemoglobin provokes strong changes in the visible absorption spectrum of the protein. The stoichiometry of this reaction, determined by spectrophotometry, is one mole of IHP per tetramer. The equilibrium dissociation constant of IHP for Hb NO , in 0·1 m -2,2-bis (hydroxymethyl)-2,2′,2″-nitrilotriethanol (pH 6·8) is 1·3×10 −6 m and increases with ionic strength and pH. IHP has only a small effect on bis ( N -maleimidomethyl) ether hemoglobin. The spectral changes induced by IHP on hemoglobin nitrosyl hybrids α 2 CO β 2 NO and α 2 NO β 2 CO have been determined. Only the latter exhibits large changes in absorbance. Comparison between the Δ∈ values found for Hb NO and α 2 NO β 2 CO suggests that in Hb NO , the α NO subunit alone contributes to the spectral changes. IHP modifies also the ultraviolet absorption spectrum of Hb NO , and decreases the reactivity of cysteine β93 toward p -chloromercurybenzoate. These changes are qualitatively similar to those that appear on deoxygenation of oxyhemoglobin, and on binding IHP to aquomethemoglobin, as previously documented by Perutz et al. (1974 a ) . The suggestion that IHP induces a new quaternary conformation in Hb NO with deoxy character is also supported by the finding that spectral changes exactly opposite to those described in the reaction of IHP with Hb NO are induced in the absence of IHP after fixation of a ligand to α 2 NO β 2 deoxy hybrid, a reaction that probably consists of a change in the structure of this hybrid from a deoxy to an oxy conformation. The results are discussed in terms of the stereochemical hypothesis suggested by Perutz et al. (1974 b ) , according to which the allosteric equilibrium constant L in hemoglobin is governed by the Fe−N 8His distance. Kinetic studies have shown that the optical changes in Hb NO and α 2 NO β 2 deoxy hybrid are slow and complex, involving Hb NO dimers and tetramers. First and indirect estimates of the tetramer-dimer equilibrium constant of Hb NO are given. They are K 4/2 =4·2×10 −6 m and 20×10 −6 m , respectively, in 0·1 m -phosphate and 0·05 m -2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol buffers (pH 6·8). It is also demonstrated that the IHP-induced spectral changes in Hb NO owe their origin to a slow rearrangement of the α NO subunit inside a quaternary structure which has already changed in conformation, as suggested by the criterion of cysteine β93 reactivity. Evidence is provided that artificially prepared α 2 NO β 2 deoxy hybrid differs some-what in conformation from naturally occurring hemoglobin intermediates, as was suggested already for valency hybrids by Cassoly & Gibson (1972) .