Biomedical subjects

B Bohn

Publications and source records attributed to B Bohn.

At least 19 recordsLinked to original sources

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

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↗

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 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

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↗

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

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↗

PubMed1987

Flow-cytometric studies with eleutherococcus senticosus extract as an immunomodulatory agent.

A placebo-controlled study of the effect of an Eleutherococcus senticosus extract (Eleukokk) on the immune system was performed with 36 healthy volunteers utilising quantitative multi-parameter flow cytometry with monoclonal antibodies directed against specific surface markers of human lymphocyte subsets. Volunteers in the verum group received 10 ml of an ethanolic (vincamine free) eleutherococcus senticosus preparation, 3 times daily for 4 weeks. In the placebo, the eleutherococcus extract was substituted by additional wine, resulting in identical final concentrations of ethanol in both preparations. The purpose of the double-blind study was the demonstration of possible effects on the cellular immune status, as determined by quantitative flow cytometry. The most salient feature in the verum group was a drastic increase in the absolute number of immunocompetent cells, with an especially pronounced effect on T lymphocytes, predominantly of the helper/inducer type, but also on cytotoxic and natural killer cells. In addition, a general enhancement of the activation state of T lymphocytes was observed. No side effects were observed during the trial or afterwards (observation period 6 months).

Adjuvants, Immunologic↗

PubMed1985

A comparison of established human lymphoma lines by flow cytometry: quantitation of Ricinus communis agglutinin binding and the effect of specific glycosidases.

Two established cell lines of human B-cell lymphomas derived from Burkitt lymphomas and their Epstein-Barr virus-transformed counterparts were analyzed with respect to their ability to bind the beta-galactoside-specific lectin Ricinus communis agglutinin (RCA). Native and sialidase- as well as sialidase-beta-galactosidase-treated cells were compared. The method for the quantitative determination of average numbers of binding sites and of apparent affinity constants was flow cytometry with fluorescence-labeled lectin. Although with native cells there was no significant deviation of the values for virus-transformed cells from those for the parent cells, some differences could be detected after glycosidase treatment. The general procedure of the combined application of specific glycosidases and the quantitation of sugar-specific lectin binding is recommended as a general strategy for the differentiation of cells with known or putative differences in biological functions.

Binding Sites↗

PubMed1979

Interaction of butene with human hemoglobin A.

The binding of various alkanes by proteins was recognized years ago. We have studied the effect of butene (C4H8), a short-chain aliphatic hydrocarbon, on the functional properties of human adult hemoglobin. Under 1 atm pressure (100 kPa) butene decreased the affinity of hemoglobin (Hb) for oxygen (p50) by 45% without altering the cooperativity of ligand binding. This effect was independent of pH (from 7.0 to 8.0) and of ionic strength. The changes in the affinity of hemoglobin for oxygen were dependent upon the partial pressure of butene and evoked a saturating mechanism of the binding site(s). Mathematical simulation of the curve relating p50 to the concentration of dissolved butene allowed us to calculate the apparent association constants for one single binding site KHb = 10.4 mmol-1 and KHbO2 = 1.53 mmol-1 to Hb and HbO2 respectively. The larger binding of butene by Hb was confirmed by a 25% decrease in K1, the first association constant of oxygen to the tetrameric hemoglobin. It is concluded that butene is an allosteric effector of human Hb which acts most likely through hydrophobic interactions. It is postulated that the oxygen-linked binding site may be located at the alpha 1 beta 2 interface.

Alkenes↗