Biomedical subjects

C Poyart

Publications and source records attributed to C Poyart.

At least 127 records · Page 7Linked to original sources

PubMed1989

Analysis of hemoglobin oxygen equilibrium curves. Are unique solutions possible?

As a hemoglobin tetramer is oxygenated, it converts from a form with low ligand affinity to a high-affinity structure. This allosteric transition occurs in partially liganded molecules, typically after two or three ligands are bound. As a result of the co-operative nature of the process, the populations of the partially liganded forms are low. The relative proportions and precise properties of these intermediate substrates are therefore difficult to measure and are subject to controversy. The problem is compounded by compensating effects; for example, over-estimation of the oxygen affinity of triply liganded forms will result in under-estimation of the proportion of these species. Specifically, published values for the oxygen affinity of the triply liganded species vary for identical conditions by a factor of more than 6. In analyses based on the highest affinity values, the triply liganded species virtually disappears. However, this affinity is usually calculated from the last few per cent of the oxygenation curve, and this part of the curve is extremely sensitive to the normalization of the data. We conclude that unique solutions for the ligand affinity and substrate populations may be impossible to achieve, and that unusual mechanisms based on particular combinations of parameters, therefore, should be viewed with caution.

Animals↗

PubMed1989

Hemoglobin Athens-Georgia [alpha 2 beta 2 40(C6)Arg----Lys] in association with beta 0-thalassemia in Tunisia.

We describe an Hb Athens-Georgia (Hb A-Ga)/beta 0-thalassemia compound heterozygosity, found in a Tunisian patient. Oxygen binding studies of red cell suspensions of this patient, containing approximately 95% Hb A-Ga, revealed an almost normal oxygen affinity. Nevertheless, dilute solutions of Hb A-Ga showed an increased overall oxygen affinity and decreased heme-heme interaction. This could be explained by a tetrameric hemoglobin with normal oxygen binding properties but with increased dissociation into monomers or dimers, as a consequence of a structural abnormality within the alpha 1 beta 2 interface. Such an interpretation would explain the increased oxygen affinity reported in previous studies performed on heterozygous Hb A/Hb A-Ga patients.

Hemoglobins, Abnormal↗

PubMed1989

Effect of ligand-affinity differences of human hemoglobin variants on electrophoretic behavior and their isolation and functional characterization.

A natural sulfated polysaccharide (agaropectin), contained in crude agar, can be used as a medium for electrophoretic separation of hemoglobin mutants, constituting a particular class of protein-ligand interactions. Mutations which either modify the electrostatic charge at the surface of the hemoglobin molecule or not, have been studied according to their putative interaction with the medium. Using conformational specificities of the hemoglobin molecule, we have also demonstrated that isoelectric focusing on a polyacrylamide gel in the absence of heme ligands represents a useful, convenient and rapid procedure for isolating silent Hb variants in their native form, provided that they exhibit an abnormal Bohr effect.

Calcium↗

PubMed1989

Analysis of oxygen binding by Xenopus laevis hemoglobin: implications for the Root effect.

We have measured oxygen binding properties for red cell suspensions and stripped hemolysate of Xenopus laevis (XL) in order to answer the question whether XL hemoglobin exhibits a Root effect. The present results show that under physiological conditions XL red cells do not exhibit all the criteria for a Root effect. Compared to human Hb A, stripped XL hemoglobin has a low oxygen affinity, a normal alkaline Bohr effect and a lower interaction with 2,3-diphosphoglycerate, its physiological allosteric effector in red cells. The values for the Hill coefficients are lower than those for human hemoglobin but XL Hb remains cooperative (n50 approximately 2), even at pH values below 6. Attempts to mimick some of the criteria for the Root effect were carried out in pure Hb A solution upon addition of potent allosteric effectors. This leads to low cooperativity and less than 100% oxygen saturation under room air at acidic pH. Under these conditions, mammalian or XL Hb have a maximum proton release at pH approximately 8 and an increased reverse Bohr effect. By contrast, a Root effect Hb exhibits a maximum proton release at neutral pH with the absence of reverse Bohr effect. Therefore the Root effect in fish Hb and the extreme stabilization of the T-state with effectors in mammalian Hb are not an identical phenomenon. Without crystallographic analyses of fish Hb exhibiting a Root effect, the molecular interpretation of this functional property still remains unexplained.

Animals↗

PubMed1989

Inhibition of oxygen-linked anion binding in Hb Camperdown [alpha 2 beta 2(104)(G6)Arg----Ser].

Oxygen equilibrium studies of purified Hb Camperdown [beta 104(G6) Arg----Ser] have revealed an increased oxygen affinity at acid pH, while it is decreased for pH values above 7.4. This accounts for an almost 40% reduction in the alkaline Bohr effect. The effects of chloride and organophosphate effectors on the oxygen affinity of Hb Camperdown are inhibited by 40-50%. In chloride-free Hepes buffer, Hb Camperdown exhibits a lower oxygen affinity than normal Hb A. The present results confirm the important role of the positively charged residues lining the beta 1 beta 2 interface in regulating the functional properties of hemoglobin.

2,3-Diphosphoglycerate↗

PubMed1989

Structure-function of Hb Marseille-Long Island [alpha 2 beta 2 N-methionyl-2(NA2) His----Pro].

Suspensions of red cells containing Hb Marseille-Long Island showed decreased oxygen affinity and low interaction with 2,3-diphosphoglycerate. Oxygen equilibrium studies of the purified component confirmed these abnormalities. Oxidation rate measurements of carbonmonoxy-Hb Marseille and carbonmonoxy-Hb A by ferricyanide showed an increased rate for the former, suggesting an increased dissociation constant for carbon monoxide. Nuclear Magnetic Resonance spectra in the high field region revealed small changes in the proximal region of the heme pocket. These results indicated that the mutation causes a perturbation at a distance from the mutation site.

2,3-Diphosphoglycerate↗

PubMed1989

Hemoglobin Köln occurring in association with a beta zero thalassemia: hematologic and functional consequences.

Hemoglobin (Hb) Köln-beta zero thalassemia compound heterozygosity was discovered in a young Greek patient. This gave us the unique opportunity for studying the functional properties of this unstable high-oxygen affinity hemoglobin variant in red cells containing almost pure Hb Köln. The red cells of the proposita exhibit morphological alterations and hematologic indices corresponding to the presence of an unstable Hb and beta thalassemia. Globin chain synthesis confirmed the association with a beta zero thalassemia gene. Oxygen-binding curves for these cells were biphasic, indicating the presence of both heme-saturated and of approximately 20% of non-cooperative Hb Köln. The major component exhibits an increased oxygen affinity, reduced cooperativeness, and normal alkaline Bohr effect. The 35-year-old proposita is active, has not been splenectomized, and has not been transfused in several years.

Adult↗

PubMed1988

Inhibition of membrane erythrocyte (Ca2+ + Mg2+)-ATPase by hemin.

Red blood cell lysis is a common symptom following severe or prolonged oxidative stress. Oxidative processes occur commonly in sickle cells, probably mediated through denatured hemoglobin and the accumulation of ferric hemes in the membranes. Calmodulin-stimulated (Ca2+ + Mg2+)-ATPase from sickle red cell membranes is partially inactivated (Leclerc et al. (1987) Biochim. Biophys. Acta 897, 33-40). In this study (Ca2+ + Mg2+)-ATPase activity from normal adult erythrocyte membranes was measured in the presence of hemin. We report a time- and concentration-dependent inhibition of the activity of the enzyme by hemin due to a decrease in the maximum velocity. Only a mild inhibitory effect was observed in the presence of iron-free protoporphyrin IX, indicating the catalytic influence of the iron. Experiments carried out with hemin (ferric iron) liganded with imidazole or with reduced protoheme (ferrous iron) liganded with carbon monoxide, demonstrated that the inhibition requires that hemin be capable of binding additional ligands. The inhibition was not influenced by the absence of oxygen but was prevented by addition of bovine serum albumin. Addition of butylated hydroxytoluene, a protective agent of lipid peroxidation, failed to prevent the inhibition of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. As dithiothreitol partially restores the enzyme activity, we postulated that hemin interacts with the thiol groups of the enzyme.

Ca(2+) Mg(2+)-ATPase↗

PubMed1988

Increased oxygen affinity with normal heterotropic effects in hemoglobin Loire [alpha 88(F9)Ala----Ser].

Increased homotropic allosteric effect, while maintaining normal heterotropic effects, was observed in hemoglobin Loire. The oxygen binding curves, at equilibrium, and the kinetic measurements demonstrated that the substitution of alpha 88(F9) Ala for a Ser results in increased oxygen affinity and decreased n50 value. The function of the residues involved in the Bohr effect or in the regulation by 2,3-bisphosphoglycerate is not altered. The effects of bezafibrate, which binds specifically to the alpha chains, was similar to that observed in Hb A. The functional properties of Hb Loire may be explained by a slight displacement of some key residues of the C-terminal region of the alpha chain destabilizing the T structure.

2,3-Diphosphoglycerate↗

PubMed1988

Photophysics and reactivity of heme proteins: a femtosecond absorption study of hemoglobin, myoglobin, and protoheme.

On the basis of our time-resolved absorption measurements of hemoglobin (Hb), myoglobin (Mb), and protoheme (PTH), either unligated or ligated with CO, O2, or NO, we propose a description of the photophysics of heme proteins that encompasses their photodissociation, the origin and fate of the observed short-lived transients, and the appearance of the ground-state, unligated heme proteins. Two distinct species are formed upon ligand photodissociation, which occurs in less than 50 fs. We assign these species to excited states of the unligated heme and label them (for the case of hemoglobin) as Hb*I and Hb*II. We suggest that Hb*I is already at least partially domed and has a spin state of at least S = 1. Hb*I decays in 300 fs to the ground-state unligated heme species, which we consider to be S = 2 and at least partially domed. The population of Hb*II varies with the ligand. It is more significant when the ligand is O2 or NO than when the ligand is CO. The similarities of the picosecond and femtosecond bleaching and absorption kinetics of HbCO with those of PTHCO (and of HbNO with those of PTHNO) indicate that in this time domain the importance of steric features of the protein are less important than the nature of the ligand itself in the geminate recombination process as well as in the relative amounts of the two heme excited states created. It is suggested that the quantum yield of ligand photodissociation is unity whether the ligand is O2, NO, or CO. The low yield of photodissociated heme-O2 or heme-NO compounds as measured on the microsecond time scale is thus attributed to a fast (2.5 ps) recombination of O2 or NO with Hb*II. We discuss geminate recombination measurements of cyanomet hybrid hemoglobins with NO and consider these results in terms of alpha and beta subunit heterogeneity. The first picosecond transient absorption spectra of cyanomet-CO hybrid hemoglobins are presented and are compared with the spectra of other heme compounds. The superimposability of the transient spectra on the equilibrium spectra of heme compounds that exhibit minimal or no cooperativity is noted and is compared with the case of cooperative systems where the transient spectra are distorted with respect to the equilibrium spectra. This distortion is interpreted in terms of an interaction of a domed heme with the F helix.

Heme↗

PubMed1988

T-state hemoglobin with four ligands bound.

Flash photolysis kinetics have been measured for ligand recombination to hemoglobin (Hb) in the presence of two effectors: bezafibrate (Bzf) and inositol hexakisphosphate (IHP). The combined influence of the two independent effectors leads to predominantly T-state behavior. Samples equilibrated with 0.1 atm of CO are fully saturated, yet after photodissociation they show only T-state bimolecular recombination rates at all photolysis levels; this indicates that the allosteric transition from R to T occurs before CO rebinding and that the allosteric equilibrium favors the T-state tetramer with up to three ligands bound. Since all four ligands bind at the rate characteristic for the T-state, the return transition from T to R must occur after the fourth ligand was bound. At 1 atm of CO, rebinding to the initial R state competes with the allosteric transition resulting in a certain fraction of CO bound at the rate characteristic for the R state; this fraction is greater the smaller the percentage dissociation. Under 1 atm of oxygen, samples are not more than 93% saturated and show mainly T-state kinetics. The results show that all four hemes can bind oxygen or CO ligands in the T structure. The fraction of the kinetics occurring as geminate is less for partially liganded (T-state) samples than for fully liganded (R-state) Hb.

Allosteric Site↗

PubMed1988

Analysis of proton release in oxygen binding by hemoglobin: implications for the cooperative mechanism.

The relationship in hemoglobin between cooperativity (dependence of the Hill constant on pH0 and the Bohr effect (dependence of the mean oxygen affinity on pH) can be described by a statistical thermodynamic model [Szabo, A., & Karplus, M. (1972) J. Mol. Biol. 72, 163-197; Lee, A., & Karplus, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7055-759]. In this model, salt bridges and other interactions serve to couple tertiary and quaternary structural changes. To test and refine the model, it is applied to the analysis of the pH dependence of the tetramer Adair constants corrected for statistical factors (K4i', i = 1-4). Attention is focused on the proton release of the first (delta H1+ = alpha log K41'/alpha pH) and last (delta H4+ = alpha log K44'/alpha pH) oxygenation steps, where K4i' are the Adair constants corrected for statistical factors. Measurements of delta H1+ and delta H4+ under carefully controlled conditions are reported, and good agreement between the model calculation and these experimental results is obtained. The salt bridges are found to be partially coupled to the ligation state in the deoxy quaternary structure; it is shown that a Monod-Wyman-Changeux-type model, in which the salt bridges are coupled only to quaternary structural change, is inconsistent with the data for delta H1. The significance of the present analysis for an evaluation of the Perutz mechanism [Perutz, M.F. (1970) Nature (London) 228, 726-734, 734-739] and other models for hemoglobin cooperativity is discussed.

Humans↗

PubMed1988

Hemoglobin J Guantanamo [alpha 2 beta 2 128 (H6) Ala----Asp] in association with hemoglobin C and alpha-thalassemia in a family from Benin.

Hemoglobin J (HbJ), Guantanamo, which had been described but once in the literature, was found in a family originating from Benin; this second case was found to be in association with hemoglobin C (HbC) and alpha-thalassemia. High-performance liquid chromatography (HPLC) procedures and microsequencing were used for characterization of the aminoacid substitution. The main hematological disorder, in relation with the instability of Hb J Guantanamo, seems to be a worsening of the rheological properties of the red blood cells (RBC), as demonstrated by ektacytometric studies. Oxygen-binding properties of the RBC were almost normal, but a slight decrease in cooperativity and lowered Bohr and 2,3-diphosphoglycerate (DPG) effects were observed for pure stripped Hb J Guantanamo. The expression of the electrophoretic charge difference was partly masked, as is often observed when the structural abnormality is situated in or near a contact area.

Amino Acid Sequence↗

PubMed1988

Functional properties of hemoglobin in human red cells: II. Determination of the Bohr effect.

Parameters of the Bohr effect (delta log P50/delta pHi) for human normal red blood cell suspensions have been calculated between intracellular pH (pHi) 5.5 and 8.5, in order to test how these quantities compare with those measured in dilute human hemoglobin solution (Hb). For a precise comparison between red cell and Hb solutions data, the cells were fractionated to reduce cellular heterogeneity and depleted of their 2,3-diphosphoglycerate (DPG) content. The P50 values were related to pHi. From the value of the Donnan equilibrium factor rH+ and the cellular water content at each extracellular pH (pHe), the variations of the intracellular chloride concentration were calculated. This quantity exhibited a three fold change between pHi 5.5 and 8.5, with extracellular chloride concentration [Cl-]e fixed at 140 mM. In the absence of DPG, chloride is the main allosteric effector of Hb and increases the alkaline Bohr effect. When all these interacting factors are accounted for, the oxygen affinity and Bohr parameters for red cell suspensions become identical to those observed for dilute Hb solutions. These results indicate that the hydration of the Hb molecules in the highly concentrated red cell milieu is not much different from that of nearly ideal solutions.

2,3-Diphosphoglycerate↗

PubMed1988

Hemoglobin Saverne: a new variant with elongated beta chains: structural and functional properties.

A 26-year-old French woman born in Saverne (France) was found to have Heinz body hemolytic anemia. Isoelectrofocusing showed the presence of an abnormal band amounting to 35% of the total hemoglobin concentration, suggesting a beta variant. Structural analysis of the abnormal beta chain showed an elongated C-terminal segment. Histidine 143 is replaced by a proline and the C-terminal sequence is identical to the corresponding segment of Hb Cranston. This new variant, named Hb Saverne, has beta chains composed of 156 amino acid residues. Studies of its functional properties showed that Hb Saverne is an unstable, high affinity variant with low cooperativity.

Adult↗

PubMed1987

Time-resolved Raman spectroscopy with subpicosecond resolution: vibrational cooling and delocalization of strain energy in photodissociated (carbonmonoxy)hemoglobin.

A Raman spectrometer that provides both subpicosecond resolution and independent, tunable pump and probe pulses is described. The spectrometer is employed to obtain time-resolved spectra of (carbonmonoxy)hemoglobin (HbCO) at times from 0.2 to 95 ps subsequent to ligand photodissociation. The spectra are interpreted in terms of a vibrationally hot heme that cools substantially in 10 ps. Concomitant with the proposed vibrational cooling is a slower relaxation, which we suggest results from a protein response to heme doming induced by ligand detachment. Results and interpretations are discussed in the context of current models of the heme photophysics and of hemoglobin reactivity.

Adult↗

PubMed1987

An expanded two-state allosteric model for interactions of human hemoglobin A with nonsaturating concentrations of 2,3-diphosphoglycerate.

Oxygen binding curves (OEC) for red cell suspensions have a biphasic shape and reduced n50 values when the concentration of 2,3-diphosphoglycerate (DPG) is lowered by aging or experimental procedures. The mechanism for the abnormal shape of the OEC has been related to variations in the activity of free DPG. DPG binds to tetrameric Hb at a single site, and in red cells its normal concentration is equivalent to that of tetrameric Hb. This equivalence renders the oxygen affinity of Hb and the shape of the OEC very sensitive to small changes in the activity of DPG. The OEC for stripped Hb solutions in the presence of nonsaturating concentrations of DPG also exhibit a biphasic shape but with much larger changes in the n values than observed for red cells. Upon addition of chloride, a known competitor of DPG binding to Hb, the shape of the OEC becomes similar to that of red cell suspensions with the same DPG/Hb ratio. Studies on Hb solutions in the presence of varying concentrations of DPG, but without chloride, have revealed that the cofactor shifts the entire OEC to the right, including both its upper and lower asymptotes. This finding indicates that DPG lowers the intrinsic oxygen affinity for both the T and R states. Theoretical considerations leading to a successful modeling of OEC obtained under varying conditions of DPG and chloride require an expanded two-state allosteric model in which allowance is made for DPG-dependent variations in the dissociation constants of oxygen for both the T and R conformations.

2,3-Diphosphoglycerate↗

PubMed1987

The calmodulin-stimulated (Ca2+ + Mg2+)-ATPase in hemoglobin S erythrocyte membranes: effects of sickling and oxidative agents.

A decrease in the reactivity of erythrocyte membrane (Ca2+ + Mg2+)-ATPase to calmodulin stimulation has been observed in aging red cells and in various types of hemolytic anemias, particularly in sickle red cell membranes. Unlike the aging process, the defect in the (Ca2+ + Mg2+)-ATPase from SS red blood cells is not secondary to a decrease in calmodulin activity and is already present in the least dense SS red blood cells separated on a discontinuous density gradient. Deoxygenated AS red cells were forced to sickle by lowering the pH, raising the osmolarity of the buffer (sickling pulse). Under these conditions an inhibition of the calmodulin-stimulated enzyme was observed only if several cycles of oxygenation/deoxygenation were applied. No alteration of the enzyme could be detected after submitting AS red blood cells to other conditions or in AA red blood cells submitted to the same treatments. This suggests that oxidative processes are involved in the alterations of the (Ca2+ + Mg2+)-ATPase activity. Treatment of membranes from AA erythrocytes by thiol group reagents and malondialdehyde, a by-product of auto-oxidation of membrane unsaturated lipids and a cross-linking agent of cytoskeletal proteins, led to a partial inhibition of the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. We postulate that the hyperproduction of free radicals described in the SS red blood cells and involved in the destabilization of the membrane may be also responsible for the (Ca2+ + Mg2+)-ATPase failure.

Anemia, Sickle Cell↗