Biomedical subjects

J Kister

Publications and source records attributed to J Kister.

At least 73 records · Page 4Linked to original sources

PubMed1994

Functional properties of beta(NA1)Val-deleted,(NA2)His-->Met hemoglobin synthesized in Escherichia coli.

Bovine Hb (hemoglobin) has a low oxygen affinity in the absence of chloride ions and DPG. Because of the increasing interest of this Hb as a potential blood substitute we have engineered a human Hb mutant with the aim of mimicking the functional properties of bovine Hb. This was achieved by deleting residue beta NA1 Val and substituting a methionine for histidine at the beta NA2 position as previously suggested by Perutz and Imai in 1980. Our results show that the artificial mutant exhibits some of the characteristics of bovine Hb but does not show the low oxygen affinity which is measured in bovine blood.

Animals↗

PubMed1993

Hb Villaverde [beta 89 (F5) Ser-->Thr]: the structural modification of an intrasubunit contact is responsible for a high oxygen affinity.

Hb Villaverde [beta 89 (F5) Ser-->Thr], identified in a Spanish patient, is a new human hemoglobin variant, electrophoreticaly silent, responsible for a severe erythrocytosis. This abnormal hemoglobin displays a very high oxygen affinity and a markedly reduced cooperativity that is partly restored in the presence of IHP. Determination of the structural abnormality was achieved on a mixture of the normal and abnormal beta-chains. After isolation of the abnormal tryptic peptide by RP-HPLC, its sequence was determined by mass spectrometry. The structural abnormality disturbs the intrasubunit interaction between helices F and H and, thus, may weaken the C-terminal bonds of the deoxy conformation and the heme contacts of several hydrophobic residues. Hb Villaverde demonstrates that this intrasubunit contact between helices F and H is essential for the cohesion of the hemoglobin molecule.

Adult↗

PubMed1993

A novel allosteric mechanism in haemoglobin. Structure of bovine deoxyhaemoglobin, absence of specific chloride-binding sites and origin of the chloride-linked Bohr effect in bovine and human haemoglobin.

The structure of bovine deoxyhaemoglobin has been determined at 2.2 A resolution and refined to an R-factor of 0.193 for all 32,583 reflections, and a free R-factor of 0.249 for 1527 reflections excluded from the refinement. The structure shows no significant differences between the alpha-carbon positions of bovine and human haemoglobin, except at the N-terminal segment and the first helix (A) which are closer to the dyad symmetry axis and pushed more tightly against the rest of the beta-subunits in the bovine form. In a search for the predicted chloride-binding sites, three-dimensional data were collected from crystals suspended in 50% polyethylene glycol buffered either with 50 mM Na phosphate (pH 7.3) +/- 0.1 M NaCl or with 0.1 M Hepes (pH 7.3) +/- 0.1 M NaBr. Difference electron density maps with and without NaCl or NaBr showed no evidence of specific halide ion-binding sites. Oxygen equilibria were measured in 10 mM Hepes buffer without added NaCl, with 0.1 mM NaCl, 0.1 M NaCl + 1 mM 2,3-diphosphoglycerate, and 0.1 M NaCl + 1 mM inositol hexaphosphate. Without added chloride, P50 of stripped bovine haemoglobin was similar to that of human haemoglobin with 0.1 M NaCl. With 0.1 M NaCl it was similar to that of human haemoglobin saturated with 2,3-diphosphoglycerate. In 0.1 M NaCl neither organic phosphate significantly affected the oxygen affinity. Titration of P50 with NaCl showed delta log P50/delta log[Cl-] of bovine and human haemoglobin to be identical. Analysis of the oxygen equilibrium curves showed the low intrinsic oxygen affinity of bovine haemoglobin to be due to a larger oxygen dissociation constant from the T-structure. The influence of chloride on P50 and on the alkaline Bohr effect is the same in bovine and human haemoglobins. It is proposed that this is due to the excess positive charges in the central cavity and its widening in the transition from the R to the T-structure. The widening would allow more chloride ions to enter and neutralize the positive charges, but these ions would remain mobile and therefore do not show up as peaks of high electron density. Repulsion between excess positive charges in the central cavity raises the free energy of the T-structure relative to the R-structure, thereby raising the oxygen affinity. Conversely, entry of chloride ions on widening of the cavity reduces the free energy of the T-structure and therefore lowers the oxygen affinity.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗

PubMed1993

Hb Saint Nazaire (beta 103[G5]Phe-->Ile): a new example of polycythemia due to a hemoglobin variant with increased oxygen affinity.

Hb Saint Nazaire [beta 103 (G5) Phe-->Ile] was found in four apparently unrelated French families. The five patients carrying this hemoglobin have been detected because of a moderate erythrocytosis. The structural abnormality of Hb Saint Nazaire concerns the same residue as in Hb Heathrow [beta 103 (G5) Phe-->Leu). A comparative functional study between these two variants showed that the increase in oxygen affinity is much lower in Hb Saint Nazaire than in Hb Heathrow. The replacement of phenylalanine G5, which is localized within the heme pocket, by a leucine abolishes several contacts between the heme and the globin and leads to an environment of the heme having some similarities with that observed in myoglobin. In contrast, the replacement of G5 by an isoleucine is likely to introduce less structural modifications.

Adolescent↗

PubMed1993

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.

Allosteric Site↗

PubMed1993

Hb Val de Marne [alpha 133(H16)Ser-->Arg]: a new hemoglobin variant with moderate increase in oxygen affinity.

Hb Val de Marne [alpha 133(H16)Ser-->Arg] was found in a French family during a neonatal hemoglobinopathy screening program. The abnormal hemoglobin was found, within a few months interval, in two newborn children who were first cousins. In the children as well as in the parents carrying this hemoglobin variant, the red cell parameters were normal. Hb Val de Marne, isolated by isoelectrofocusing, displayed a slightly higher auto-oxidation rate than Hb A. Its oxygen affinity was increased 1.7-fold in comparison to that of Hb A. The heterotropic effects were normal.

Adult↗

PubMed1992

Hemoglobin Rouen (alpha-140 (HC2) Tyr-->His): alteration of the alpha-chain C-terminal region and moderate increase in oxygen affinity.

Hb Rouen (alpha 140(HC2) Tyr-->His) is a moderately high oxygen-affinity variant that was found in coincidence with polycythemia vera in a French patient. This hemoglobin provides an example of an alteration of the C-terminus of the alpha-chain, a region involved in the mechanisms of allosteric regulation. The increase in oxygen-affinity and decrease in cooperativity of this variant is much smaller than that resulting from the same substitution in the beta-chain. This model provides additional evidence for the inequivalence between the alpha- and beta-subunits.

Aged↗

PubMed1992

Allosteric modifiers of hemoglobin: 2-[4-[[(3,5-disubstituted anilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid derivatives that lower the oxygen affinity of hemoglobin in red cell suspensions, in whole blood, and in vivo in rats.

Two new potent allosteric effectors of hemoglobin, RSR-4 [2-[4-[[(3,5-dichloroanilino)carbonyl]-methyl]phenoxy]-2- methylpropionic acid] and RSR-13 [2-[4-[[(3,5-dimethlanilino)carbonyl]methyl]-phenoxy]-2-methylp rop ionic, are compared to the previously reported compounds L3,5 and L3,4,5 [Lalezari, I., Lalezari, P., Poyart, C., Marden, M., Kister, J., Bohn, B., Fermi, G., & Perutz, M. F. (1990) Biochemistry 29, 1515]. Unlike L3,5 and L3,4,5, RSR-4 and RSR-13 are less impeded by physiological concentrations of serum albumin. RSR-4 has also been shown to be more effective than L3,5 in shifting the allosteric equilibrium of bovine Hb toward the low-affinity T-state. X-ray crystal studies show that both RSR-4 and RSR-13 bind to only one pair of symmetry-related sites in the Hb central water cavity whereas previous studies on L3,5 and L3,4,5 demonstrated a second pair of symmetry-related binding sites near Arg 104 beta. Three major interactions between these allosteric effectors and Hb include the acid group with the guanidinium group of C-terminal Arg 141 alpha, the effector's amide oxygen with the ammonium ion of Lys 99 alpha, and the phi electrons of the halogenated or methylated aromatic ring and Asn 108 beta. No explanation has been found for the difference in number of binding sites observed for RSR-4 and RSR-13 (two sites) compared to L3,5 and L3,4,5 (four sites); also no correlation has been made between the number of binding sites and degree of allosteric shift in the oxygen equilibrium curve.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

Allosteric properties of haemoglobin beta 41 (C7) Phe-->Tyr: a stable, low-oxygen-affinity variant synthesized in Escherichia coli.

In human deoxy haemoglobin, the alpha 42(C7)Tyr-residue is hydrogen-bonded to beta 99(G1)Asp which stabilizes the low-oxygen-affinity deoxy conformation. We engineered a haemoglobin with Tyr for Phe at the homologous C7 position in beta-chains. The oxygen affinity of the variant is decreased about two-fold relative to Hb A while keeping similar KR and KT values. This mutant may be a candidate for the development of an artificial oxygen carrier, as it would not require an external effector for significant oxygen unloading in vivo.

Allosteric Site↗

PubMed1992

Hemoglobin Thionville. An alpha-chain variant with a substitution of a glutamate for valine at NA-1 and having an acetylated methionine NH2 terminus.

In hemoglobin (Hb) Thionville, the substitution of a glutamic acid for the alpha-chain NH2-terminal valine inhibits the cleavage of the initiator methionine which is then acetylated. The elongation of the alpha-chain NH2 terminus modifies the three-dimensional structure of hemoglobin at a region that is known to have an important role in the allosteric regulation of oxygen binding. Relative to Hb A, Hb Thionville has a lower affinity for oxygen, and the heterotropic allosteric effects of protons, chloride, and bezafibrate are reduced. In contrast, the response to 2,3-diphosphoglycerate is normal. Analysis of oxygen equilibrium data within the framework of the two-state allosteric model indicates that the structure of deoxy Hb Thionville is stabilized relative to that of deoxy Hb A. The x-ray crystal structure of deoxy Hb Thionville shows that the glutamate side chain extends away from the alpha 1-alpha 2 interface, whereas the methionine side chain (which has two conformations) extends into the alpha 1-alpha 2 interface, physically displacing chloride and bezafibrate. The increased stability of deoxy Hb Thionville is due to new intrasubunit and intersubunit contacts made by the methionine. These interactions replace the indirect contacts, made through bound chloride ions, that Val-1 alpha normally contributes to the alpha 1-alpha 2 interface.

Acetylation↗

PubMed1992

Structure of the EF corner favors deamidation of asparaginyl residues in hemoglobin: the example of Hb La Roche-sur-Yon [beta 81 (EF5) Leu----His].

Some abnormal hemoglobins constitute models which allow one to evaluate the structural requirements for post-translational modifications in proteins, such as deamidation. Hb La Roche-sur-Yon [beta 81 (EF5) Leu----His] is an unstable hemoglobin variant displaying a moderately increased oxygen affinity. About half of the abnormal hemoglobin, in addition to the substitution at position beta 81, carries a deamidation of the neighboring asparagine residue, beta 80 (EF4). The histidine at position beta 81 cannot fit into the small hydrophobic pocket which normally accomodates the leucine residue. This structural change opens the heme pocket and modifies the general conformation of the EF segment, thus explaining the increase in oxygen affinity and the achievement of a three-dimensional structure favoring asparagine deamidation. Histidine beta 81 could also act as a catalyst in the deamidation reaction. Deamidation has already been reported for two other variants of the EF corner, Hb Providence [beta 82 (EF6) Lys----Asn] and Hb J Singapore [alpha 79 (EF8) Ala----Gly]. In all these cases it seems that a histidine may catalyze the deamidation of the asparagine residues and that disturbing the folding of the EF corner will provide an extra flexibility favoring the reaction.

Adolescent↗

PubMed1992

Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins.

Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb Hb S, beta 6 Glu-->Val) have been obtained from X-ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site-directed mutagenesis with expression of the Hb S beta subunits in Escherichia coli provides the experimental tools to test these models. For Hb S, the beta 6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the beta 6 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the beta 6 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the beta 23 Val-->Ile mutation at the axial contact with either the beta 6 Glu-->Val or beta 6 Glu-->Ile substitution in the same beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1992

Molecular adaptation of hemoglobin function in mammals.

Vertebrate hemoglobins are tetramers made of two pairs of alpha and beta subunits each containing a hydrophobic pocket where a heme molecule binds tightly and allows for the reversible binding of oxygen. Both tertiary and quaternary structures are ideally suited for the loading and unloading of oxygen necessary for the metabolic requirements of the organisms. Starting from a single ancestor hemoglobin subunit, evolutionary processes have led to heterologous tetramers exhibiting a moderate oxygen affinity due to heme-heme interaction (allosteric mechanism) which may be further modulated through electrostatic interactions with chloride and/or organophosphate anions present in the red cells. These effectors, which bind preferentially to the deoxy-Hb tetramers at a distance from the heme groups, play a major role in the adaptation of the respiratory properties of hemoglobin to either allometric-dependent oxygen needs or to various hypoxic environments such as altitude, burrowing, or foetal life. In most cases the existence or the strength of the effector-Hb complexes, hence the changes in the allosteric equilibrium, may be ascribed to one or a few mutations of residues at the effector binding sites. Typical examples of these mechanisms are described.

Adaptation, Physiological↗

PubMed1992

HB Al-Ain Abu Dhabi [alpha 18(A16)Gly----Asp]: a new hemoglobin variant discovered in an Emiratee family.

During a routine program of hemoglobin screening performed in the United Arab Emirates, we observed an electrophoretically fast-moving variant in a 9-month-old girl and in several members of her family. The structural determination, performed by reversed phase high performance liquid chromatography and amino acid sequencing, revealed a new variant that we named Hb Al-Ain Abu Dhabi [alpha 18(A16) Gly----Asp]. Its functional properties were normal.

Amino Acid Sequence↗

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

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↗