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C Poyart

Publications and source records attributed to C Poyart.

At least 109 records · Page 6Linked to original sources
PubMed1992

[Molecular engineering of hemoglobin for transfusion].

The search for a safe alternative to conventional blood transfusion has been directed towards either the use of synthetic perfluorochemicals or the biochemical manipulation of highly purified stroma-free Hb solutions prepared from outdated bank blood. However when using human blood, one does not eliminate the risks of viral infections. A novel source of Hb appeared with recent biotechnology techniques enabling one to synthesize recombinant Hb from microorganisms (E coli or S. cerevisiae) whose genome has been modified to contain globin genes. Normal human Hb A in solution, i.e. outside the red cells, is not suited for direct usage as a blood substitute because i) its high oxygen affinity, due to the absence of 2,3 DPG in the plasma, precludes sufficient O2 unloading to the tissues; ii) at low concentration, relative to that in the red cells, tetrameric Hb dissociates into dimers which escape the circulatory system by renal filtration or rapidly oxidize to the non functional metHb form. Expression of alpha- and beta-globins in Escherichia coli and in Saccharomyces cerevisiae enables one to introduce appropriate mutation(s) in the globin genes resulting in the expression of a synthetic Hb with low oxygen affinity, resembling that of normal whole blood; functional Hb has also been produced in a soluble form either in E. coli or in yeast. The coexpression of beta globin chains and alpha globin subunits linked by a peptide bond results in the direct synthesis of stabilized and fully functional Hb tetramers. Lastly, dilute haemoglobin solutions are prone to autooxidize and the rate of oxidation appears to be inversely proportional with the oxygen affinity of the heme groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination.

Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.

Binding Sites↗
PubMed1991

Inhibition of the erythrocyte (Ca2+ + Mg2+)-ATPase by nonheme iron.

The erythrocyte calmodulin-stimulated (Ca2+ + Mg2+)-ATPase (CaM-ATPase), an integral membrane protein, is inhibited in different types of congenital hemolytic anemias for which oxidative processes appear as a common feature. The oxidation of hemoglobin and its degradation lead to the accumulation of ferric heme (hemin) and nonheme iron in the red cell. We have shown previously that hemin inhibits the activity of the enzyme of normal erythrocyte (Leclerc et al. (1988) Biochim. Biophys. Acta, 946, 49-56) involving an oxidation of thiol groups. The present study demonstrates that nonheme iron also inhibits the CaM-ATPase activity. In contrast with hemin, the inhibition of the enzyme induced by the nonheme treatment is prevented by butylated hydroxytoluene, a protecting agent of unsaturated phospholipid peroxidations, while dithiothreitol, a reducing agent of protein disulfide bridges, does not restore the activity of the enzyme. We conclude that nonheme iron inhibits the enzyme at least in part, through the peroxidation of phospholipids of the membrane bilayer.

Butylated Hydroxytoluene↗
PubMed1991

Allosteric transition in triply met-haemoglobin.

Methaemoglobin undergoes a transition to a T-like form at acid pH in the presence of strong effectors such as inositol hexakisphosphate (IHP), as evidenced by spectroscopic and oxidation potential measurements. Since oxygen and CO do not bind to the ferric haems, it is difficult to compare the properties of the R-met and T-met forms with those of ferrous haemoglobin. We have therefore prepared 90% oxidized samples, where the dominant signal for ligand (oxygen or CO) binding is due to tetramers with three met haems. Measurements were made of the oxygen equilibrium curves and CO rebinding kinetics after photodissociation. Without effectors, the partially oxidized samples show mainly R-state properties. Addition of IHP at acid pH induces an increase in T-state behaviour, as indicated by a lower oxygen affinity and a higher fraction of the slow bimolecular component for CO rebinding.

Allosteric Regulation↗
PubMed1991

Hemoglobin Redondo [beta 92(F8) His----Asn]: an unstable hemoglobin variant associated with heme loss which occurs in two forms.

Hb Redondo [beta 92(F8) His----Asn] illustrates how post-translational structural modifications may modify the phenotypic expression of an unstable hemoglobin. This variant was found in a Portuguese patient suffering from a chronic hemolytic anemia. The electrophoretic pattern demonstrated that it occurred in two forms, both being semi-hemoglobins: the fastest one migrating like HbS and the other like HbA2; after a few days of storage at 4 degrees C the intensity of the slowest Hb fraction decreased while that of the other increased proportionally. In both cases, the RP-HPLC analysis of the tryptic digest of the aminoethylated beta chains demonstrated the presence of an abnormal beta T10 peptide carrying a His----Asx substitution. Microsequence study of these two peptides demonstrated that the slowest abnormal Hb fraction had a beta 92 His----Asn substitution and the fastest a His----Asp at the same site. All these results suggest that the beta 92 His----Asn variant loses readily its heme group and that a deamidation occurs rapidly in vitro, yielding a beta 92 Asp semi-hemoglobin. The oxygen affinity of the patient's red blood cells was increased, leading to a stimulation of erythropoiesis and to a macrocytic hemolytic disease.

Amino Acid Sequence↗
PubMed1991

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.

Allosteric Regulation↗
PubMed1990

Hemoglobin Saint Mandé [beta 102 (G4) Asn----Tyr]. Functional studies and structural modeling reveal an altered T state.

Oxygen equilibrium studies of purified hemoglobin Saint Mandé (Hb SM) [beta 102 (G4) Asn----Tyr] reveal a decreased oxygen affinity and cooperativity but to a lesser extent than found for Hb Kansas (beta 102 Thr). The low affinity of Hb SM depends on environmental conditions: eliminating chloride or raising the pH greatly elevated the ratio of p50 of Hb SM to that of Hb A. The alkaline Bohr effect is reduced by about 40%. The effects of anions (chloride, organophosphates) binding to deoxy Hb SM are also reduced. These data indicate that the functional properties of Hb SM are intermediary between Hb A and Hb Kansas. In addition, molecular graphics modeling of Hb SM in the oxy and deoxy structures indicate the possibility of a new hydrogen bond in the T state between beta(1)102 Tyr and alpha(2)42 Tyr. Stabilisation of the T state in this manner is a plausible explanation for several of the effects observed.

Asparagine↗
PubMed1990

Changes of polymerization and conformation of hemoglobin S induced by thiol reagents.

Thiol reagents, covalently bound to cysteine beta 93, either inhibit or facilitate the polymerization process of hemoglobin S. The progelling effect of parahydroxymercurybenzoate or 2,2'-dithiodipyridine contrasted with the increased oxygen affinity and the destabilization of the T state of Hb shown by functional and NMR studies. Thiol reagents increased the oxygen affinity of Hb from 30 to 1000%. Such variability was also observed in the reduction (up to 50%) of the alkaline Bohr effect. We show that the antigelling or progelling activity of thiol reagents does not depend solely on the concentration of molecules present in the deoxy T state but that specific effects of the reagent affects molecular interactions of the hemoglobin S polymerization process.

2,2'-Dipyridyl↗
PubMed1990

Hemoglobin Calais [beta 76 (E20) Ala----Pro]: a hemoglobin variant with decreased intrinsic oxygen affinity.

Hb Calais [beta 76 (E20) Ala----Pro] is a new human hemoglobin variant displaying a decreased oxygen affinity. The only electrophoretical difference with Hb A was a slightly more acidic isoelectric point. A 2-fold decrease in the oxygen affinity was found by equilibrium measurements performed in a suspension of intact red blood cells and in the lysate. It was confirmed by kinetic studies of the purified abnormal hemoglobin. The rate of methemoglobin formation at 37 degrees C of Hb Calais was also increased relative to Hb A. The mechanism by which the Pro for Ala substitution of an external residue in the beta-chains results in these profound functional abnormalities is unclear. Subtle changes at the heme pocket, at a distance from the mutation, may be a plausible explanation for the effects observed.

Adult↗
PubMed1990

Heme-CO as a probe of the conformational state of calmodulin.

The interaction of heme-CO with calmodulin, in the presence of calcium, leads to a complex of four heme-CO molecules per protein. No interaction was observed in the absence of calcium. The binding of heme-CO to calmodulin was monitored by the shift in the Soret absorption band from 407 to 420 nm (bound form); the four sites are not spectrally identical. The ligand CO can be photodissociated from the calmodulin-heme-CO complex and the biomolecular recombination kinetics also indicate a heterogeneous mixture. The complex does not bind oxygen reversibly. As calmodulin has only one histidine, the hemes are apparently not bound by the iron atom as in hemoglobin, but are probably loosely associated (Kd = 0.5 microM) in hydrophobic pockets which apparently open when the protein is activated by calcium.

Animals↗
PubMed1990

Fitting abnormal oxygen equilibrium curves of hemoglobin.

A growing number of oxygen equilibrium curves for hemoglobin (Hb) mutants, post-translational modifications, or the binding of potent new effectors of Hb cannot be fitted adequately with the two-state model. Examples are curves showing double maxima in the derivative of the Hill plot, or slopes of less than unity. We present such examples of modified hemoglobins and strong effectors in this study and calculate at which substate level the two-state model differs from the data. Analysis of hemoglobin oxygen equilibrium curves is reconsidered using the two-state model extended to allow variation of the individual substate probabilities. In this way the effect on the equilibrium due to perturbations in energy of each substate can be studied as a diagnostic tool.

Allosteric Regulation↗
PubMed1990

Structure and function of hemoglobin variants at an internal hydrophobic site: consequences of mutations at the beta 27 (B9) position.

We have studied the structure-function relationships in newly discovered hemoglobin (Hb) mutants with substitutions occurring at the tight and highly hydrophobic cluster between the B and G helices in the beta chains, namely, Hb Knossos or beta A27S and Hb Grange-Blanche or beta A27V. The beta A27S mutant has a 50% decrease in oxygen affinity relative to native human Hb A, while the beta A27V mutant has an increased oxygen affinity. We have also engineered the artificial beta A27T mutation through site-directed mutagenesis. This new mutant exhibits functional properties similar to those of Hb A. None of these mutants is unstable. X-ray analyses show that the substitution of Val for Ala may reduce the relative stability of the T structure of the molecule through packing effects in the beta chains; for the beta A27S mutant a new hydrogen bond between serine and the carbonyl O at beta 23 (B5) Val is observed and is likely to increase the relative stability of the T structure in the mutant hemoglobin. However, no significant changes in the crystals were observed for these mutants between the quaternary R and T structures relative to native Hb A. We conclude that small tertiary structural changes in the tight hydrophobic B-G helix interface are sufficient to induce functional abnormalities resulting in either low or high intrinsic oxygen affinities.

Binding Sites↗
PubMed1990

A new hemoglobin variant found during investigations of diabetes mellitus: Hb Pavie [alpha 135 (H18) Val----Glu].

Hb Pavie [alpha 135 (H18) Val----Glu], found during HbA1c measurement in a patient of Italian origin investigated for diabetes mellitus, exemplifies how the presence of an abnormal hemoglobin interferes with the measurement of glycated hemoglobin. This variant hemoglobin migrates as Hb A1c on polyacrylamide gel isoelectric focusing (IEF) and therefore hindered the estimation of glycated hemoglobin by this method. By ion-exchange high-performance liquid chromatography (IE-HPLC) Hb Pavie was eluted as a shoulder of the major component and the corresponding glycated fraction together with Hb A1b. Hb Pavie was purified in order to determine how its functional properties may modify red cell survival. The only functional abnormality observed was a slight decrease of the oxygen affinity, and therefore the total amount of glycated hemoglobin was not expected to be decreased by a shortening of the red cell survival.

Amino Acid Sequence↗
PubMed1990

New effectors of human hemoglobin: structure and function.

We describe the actions of two new allosteric effectors of hemoglobin, 2-[4-(3,5-dichlorophenylureido)phenoxy]-2-methylpropionic acid (L35) and 2-[4-(3,4,5-trichlorophenylureido)phenoxy]-2-methylpropionic acid (L345). Each of them binds to two pairs of symmetry-related sites in the central cavity of human deoxyhemoglobin. One pair of sites overlaps with that occupied by bezafibrate [Perutz et al. (1986) J. Am. Chem. Soc. 108, 1064-1078]. The other sites are new, and the pair occupied by L35 is different from that occupied by L345. All the sites are at least 20 A from the site where organic phosphates are bound. L345 is by far the most potent allosteric effector of hemoglobin ever described. At a concentration of 0.1 mM, it raises the P50 of a suspension of red cells by 50%; at 0.2 mM it raises the P50 2.5-fold. At acid pH, it reduces Hill's coefficient to near unity and prevents complete oxygen saturation even under 1 atm of pure oxygen. In azidemethemoglobin at pH 6, it induces a transition to higher spin. These properties are reminiscent of those of teleost fish hemoglobins that exhibit a Root effect. The influence of L35 and L345 and that of organic phosphates on the oxygen affinity are additive, but they compete with chloride. L35 acts more weakly than L345, but can be made to induce the same effects as L345 alone by adding inositol hexaphosphate. Both compounds increase the alkaline and acid Bohr effects. They alter the bimolecular kinetics of CO recombination after a flash by increasing the slowly reacting fraction of hemoglobin in the T state at the expense of the fast-reacting fraction in the R state.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1990

Effectors of hemoglobin. Separation of allosteric and affinity factors.

The relative contributions of the allosteric and affinity factors toward the change in p50 have been calculated for a series of effectors of hemoglobin (Hb). Shifts in the ligand affinity of deoxy Hb and the values for 50% ligand saturation (p50) were obtained from oxygen equilibrium data. Because the high-affinity parameters (liganded conformation) are poorly determined from the equilibrium curves, they were determined from kinetic measurements of the association and dissociation rates with CO as ligand. The CO on-rates were obtained by flash photolysis measurements. The off-rates were determined from the rate of oxidation of HbCO by ferricyanide, or by replacement of CO with NO. The partition function of fully liganded hemoglobin for oxygen and CO is only slightly changed by the effectors. Measurements were made in the presence of the effectors 2,3-diphosphoglycerate (DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), and two recently synthesized derivatives of Bzf (LR16 and L35). Values of p50 change by over a factor of 60; the on-rates decrease by nearly a factor of 8, with little change in the off-rates for the liganded conformation. The data indicate that both allosteric and affinity parameters are changed by the effectors; the changes in ligand affinity represent the larger contribution toward shifts in p50.

Allosteric Site↗
PubMed1990

Enhanced polymerization of recombinant human deoxyhemoglobin beta 6 Glu----Ile.

Polymerization of the deoxy form of sickle cell hemoglobin (Hb S; beta 6 Glu----Val) involves both hydrophobic and electrostatic intermolecular contacts. These interactions drive the mutated molecules into long fibrous rods composed of seven pairs of strands. X-ray crystallography of Hb S and electron microscopy image reconstruction of the fibers have revealed the remarkable complementarity between one of the beta 6 valines of each molecule (the donor site) and an acceptor site at the EF corner of a neighboring tetramer. This interaction constitutes the major lateral contact between the two strands in a pair. To estimate the relative importance of this key hydrophobic contact in polymer formation we have generated a polymerizing Hb with isoleucine at the beta 6 position (beta E6I) by site-directed mutagenesis. The mutated beta chains were produced in Escherichia coli and reassembled into functional tetramers with native alpha chains. Compared to native Hb S, the beta E6I mutant polymerizes faster and with a shortened delay time in 1.8 M phosphate buffer, indicating an increased stability of the nuclei preceding fiber growth. The solubility of the beta E6I mutant Hb is half that of native Hb S. Computer modeling of the donor-acceptor interaction shows that the presence of an isoleucine side chain at the donor site induces increased contacts with the receptor site and an increased buried surface area, in agreement with the higher hydrophobicity of the isoleucine residue. The agreement between the predicted and experimental differences in solubility suggests that the transfer of the beta 6 valine or isoleucine side chain from water to a hydrophobic environment is sufficient to explain the observations.

Base Sequence↗
PubMed1990

Haemoglobin alpha 2 beta 2 23Val----Ile produced in Escherichia coli facilitates Hb S polymerization.

The doubly substituted variant Hb S-Antilles (beta 6 Glu----Val, beta 23 Val----Ile) produces sickling in heterozygous carriers. The Csat value for pure deoxyHb S-Antilles is nearly half that of deoxyHb S. Dilute solutions of pure Hb S-Antilles have a lower oxygen affinity than those of Hb A or Hb S. The mutant Hb alpha 2 beta 2 23 Val----Ile was synthesized in E. coli. It exhibits a decreased oxygen affinity compared to Hb A and does not polymerize in 1.8 M phosphate buffer. Mixtures of equal amounts of Hb S + Hb beta 23 Val----Ile have a decreased Csat value compared to mixtures of Hb S + Hb A. The beta 23 Val in Hb S contributes to the axial contact joining molecules in each single filament. Substituting Ile for Val at this site increases the strength of this contact through hydrophobic interactions, allowing increased stability of the lateral contact between filaments in pair, which is the specific unit structure of polymers in deoxyHb S.

Escherichia coli↗