Biomedical subjects

B Bohn

Publications and source records attributed to B Bohn.

At least 37 records · Page 2Linked to original sources

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

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

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↗

PubMed1987

Functional properties of hemoglobin in human red cells. I. Oxygen equilibrium curves and DPG binding.

Oxygen equilibrium curves (OEC) for human red blood cells (RBC) have revealed a large asymmetry with an nmax value reached at approximately 90% saturation. This contrasts with the symmetrical shape of the OEC for diluted HbA solutions. We prepared resealed red blood cells (RRBC) through controlled hemolysis and resealing in order to obtain a low Hb concentration (approximately equal to 60 g/L) and DPG/Hb4 molar ratio ranging from 0.2 to 4. When the DPG/Hb4 was close to unity, OEC still showed a large asymmetry. On the contrary, the OEC became symmetrical when DPG/Hb4 was either negligible or 3-4 fold the normal value. The asymmetrical OEC for intact RBC can be fitted with a theoretical model taking into account the variations of free DPG concentration during oxygenation. We conclude that the asymmetrical shape of OEC observed in intact fresh RBC is not due to the high protein concentration present in the RBC but to a functional heterogeneity of Hb due to the changes of the free 2,3-diphosphoglycerate (DPG) concentration during oxygenation.

2,3-Diphosphoglycerate↗

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↗

PubMed1987

Biochemical characterization and immunomodulatory action of thymic components as determined by flow cytometry on human lymphocytes.

A commercial preparation from calf thymus was separated into its components, then chemically characterized and tested for immunomodulatory efficiency. For the latter, a novel in vitro test system is described as a relatively simple screening procedure. It employs whole blood cultures and flow cytometry with fluorescent monoclonal antibodies defining human T-cell subsets. As a reference substance, thymosin fraction 5 was used. It could be shown that the effects of the entire thymic preparation as well as of one of its peptidic components resembled closely the ones obtained with thymosin fraction 5. In several cases, i.e. with donors relatively deficient in suppressor/cytotoxic T-lymphocytes, the effect of the former was even superior to that of thymosin fraction 5.

Antibodies, Monoclonal↗

PubMed1986

Isolation of human haemoglobin variants with altered Bohr effect. Application to haemoglobin Rainier.

Isoelectric focusing on polyacrylamide gel in the absence of haem ligands represents a useful, convenient and rapid procedure to isolate silent Hb variants in their native forms, provided that they exhibit an abnormal Bohr effect. The amount of material which is eluted is sufficient for both a limited functional study and a structural determination using microscale high-performance liquid chromatography. This is exemplified by the isolation and the study of Hb Rainier.

Chromatography, High Pressure Liquid↗

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↗

PubMed1985

A new case of hemoglobin Providence (alpha 2 beta 2 82 (EF6) Lys----Asn or Asp) discovered in a French Caucasian family. Structural and functional studies.

A new case of Hb Providence was discovered in a French caucasian family presenting a mild polycythemia. Structural and functional studies of the two abnormal fractions (Hb Providence Asn and Asp) have been performed. These confirm the abnormal characteristics of Hb Providence described previously. Red cells containing Hb Providence were fractionated with a Percoll Albumin density gradient. The respective amounts of the two components were determined in the youngest and oldest cells. We observed a slight increase of Hb Providence Asp from 34 to 37% during the life span of the erythrocytes which confirms that the deamidation of Hb Providence Asn to Hb Providence Asp is a fast process, already present and close to its maximum, in the reticulocytes. Both abnormal components are stable in the presence of isopropanol.

Adolescent↗

PubMed1985

[Simple and rapid method for the fractionation of red blood cells in sickle cell anemia].

The density of normal red blood cells increases during their 3-month life span. This characteristic has been used for many years to separate younger from older cells by centrifugation, allowing biochemical and physiological studies on the ageing process in red cells to be performed. The methods which have been proposed to date using discontinuous density gradients made of high density chemical compounds required high speed centrifugation of long duration. These gradients with pre-established density layers are well designed to separate red cells when their density varies over a small range as in blood from healthy donors. Pathological red cells such as found in sickle cell anaemia, thalassaemia syndromes or haemolytic anaemia of various origins display a wide and unpredictable range of densities which precludes the use of a standard discontinuous gradient. Because the fractionation of such pathological blood specimens is of great clinical and biochemical interest we report the details of a new method which combines two steps: firstly and analytical method performed with phthalate esters which allows estimation of the distribution of particular red cells, secondly a preparative method using Percoll solutions to prepare the discontinuous density gradient according to the histogram given by the first step. Two examples are described illustrating this interesting method which may possibly be applied to other blood disorders.

Anemia, Sickle Cell↗

PubMed1984

Structure and function of Hb Saint-Jacques (alpha 2 beta 2 140 (H18) Ala----Thr): a new high-oxygen-affinity variant with altered bisphosphoglycerate binding.

A low P50 value in a fresh red blood cell suspension was discovered in a polycythemic patient (Hb 19 g X dl-1). Routine acid and alkaline electrophoreses of the hemolysate were identical to normal hemolysate. Isoelectrofocusing (pH gradient 6-8) did not reveal any abnormal band whether performed with the fully liganded or deoxygenated samples. Precise analyses of the oxygen dissociation curves of the propositus' red cells demonstrated a biphasic Hill plot, a normal Bohr effect and low interaction with 2,3-bisphosphoglycerate (2,3-DPG). Studies on the unfractionated hemolysate confirmed these observations and the inhibition of the effect of organic phosphates. Structural studies were carried out on the mixture of beta A + beta X chains and revealed the presence of two beta Tp14 peptides. Sequencing the abnormal beta Tp14 peptide showed the substitution Ala----Thr of the beta 140 (H18) residue. This new variant was named Hb Saint-Jacques. Examination of the three dimensional model of HbAo indicates that the substitution beta 140 (H18) Ala----Thr induces van der Waals interactions with the nearby lysine-82 (EF6) and leucine-81 (EF5) and a displacement of the EF corner of the beta chains. This is likely to change the normal position of the lysine-82 (EF6), a major anionic binding site in the central cavity between the two beta chains. Functional studies confirm the interpretation of a steric hindrance inhibiting the binding of large organic phosphates to Hb Saint-Jacques.

2,3-Diphosphoglycerate↗

PubMed1984

The effect of specific glycosidases on Ricinus communis agglutinin binding to cell surfaces of two tumor sublines. A comparative flow-cytometric study.

The effects of the sequential application of specific glycosidases on surfaces of living mammalian cells were studied with respect to their ability to bind the beta-galactoside-specific lectin, Ricinus communis agglutinin (RCA). Sialidase and beta-galactosidases from different sources were tested for their actions on two strains of mouse lymphoma cells differing markedly in their metastatic potential. Binding studies were performed by quantitative flow cytometry with fluorescent RCA, and numbers of specific binding sites and equilibrium association constants for the lectin on living cells were determined before and after the various enzyme treatments. Although the number of binding sites for native and sialidase-treated cells were almost identical for both cell strains, differences in the apparent affinity constants could be detected. Differences between the two strains became even more pronounced, also with respect to the number of binding sites, after treatment with beta-galactosidases from S. pneumoniae and from bovine testis. It is suggested that such combined strategies provide valuable tools for the differentiation of surface carbohydrate moieties on intact living cells, especially for comparative purposes.

Animals↗

PubMed1984

[Erythrocytosis due to a high-affinity hemoglobulin: mutant hemoglobin Saint-Jacques beta 140 (H18) Ala----Thr with a change in the 2,3-diphosphoglycerate binding site].

All the high oxygen affinity variants have substitutions in regions that are crucial to hemoglobin function: mainly the alpha 1 beta 2 interface, the C-terminal end of the beta chain and the aminoacid residues involved in the 2,3 disphophoglycerate (2,3 DPG) binding site. In this report we describe a new variant with familial erythrocytosis: hemoglobin Saint-Jacques beta 140 (H18) Ala----Thr, whose main abnormality is a defect in organic phosphate binding in the central cavity between to two beta chains. This variant could not be separated from hemoglobin A by standard electrophoretic methods or by isoelectric focusing. The abnormal peptide was isolated by reverse-phase high performance liquid chromatography and the structural modification determined by manual sequencing micro-method. Functional studies on red blood cells as well as on stripped lysate showed increased oxygen affinity, normal Bohr effect, decreased heme-heme interaction and loss of the 2,3 DPG regulatory effect.

2,3-Diphosphoglycerate↗

PubMed1982

Temperature independence of the alkaline Bohr effect in pig red cells and pig haemoglobin solutions.

The influence of temperature on the oxygen affinity and the alkaline Bohr effect of pig red cells and pig hemoglobin solutions has been compared to that of human adult red cells and human adult hemoglobin. Pig red cells and pig Hb evidence a lower affinity for oxygen in various conditions of pH, temperature and salt concentration, in the presence as well as in the absence of organic phosphates. It has been observed that the alkaline Bohr effect of pig Hb was reduced by 20-25% compared to Hb A0 and independent of changes in temperature, contrary to human Hb A0. Titrations of pig Hb with C1- indicate a lower heterotropic effect of this anion at low concentration of the salt. It is concluded that this may be the origin of the temperature independence of the alkaline Bohr effect in pig Hb. Conversely, the temperature dependence of the alkaline Bohr effect of Hb A0 should be related to the oxygen-linked binding of C1- at the alpha 1-alpha 2 interface.

Adult↗