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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.

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.

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.

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.

Functional properties of tetrameric HbA are schematically described in this short review. After considering the various structures of the protein and its binding with heme, an attempt is made to explain the influence of this structure on the affinity of Hb for oxygen, cooperative binding of oxygen, and the alkaline Bohr effect. Fixation of erythrocytic anions (chlorine and 2,3 diphosphoglycerate) modulate certain intrinsic properties, allowing the Hb molecule to provide for efficacious transport of oxygen between the environment and the metabolically active tissues.

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.

The effects of hypoxic hypoxia on renal hemodynamics and metabolism have been studied in anaesthetized mechanically ventilated rabbits. Acute hypoxa (FIO2 = 0.10, PaO2 = 35 torr) induces at constant mean arterial pressure a 45% decrease in RBF, GFR, TNa and RVO2 whereas free water clearance increases. These alterations were still apparent 50 min after resuming normal arterial oxygenation. In order to assess the role of the stimulation of catecholamine release in these observations, two other sets of experiments were performed: 1) the animals were ventilated with the same hypoxic gas mixture but after alpha adrenergic blockade (phentolamine: 0.2 mg - kg - min-1 i.v.), 2) hypoxia was induced by ventilating the animals with CO (FICO = 0.002) at constnat PaO2. Increase in renal vascular resistance and reduction of renal O2 uptake were still observed. This indicates that adrenergic stimulation cannot fully explain the renal vasoconstriction encountered in hypoxia. The role of a local vasoactive factor, especially that of the renin angiotensin system is discussed. The apparent O2 cost of Na reabsorption was not greatly modified by any type of hypoxia and the Na: O2 ratio remained close to the value observed in normoxic animals. This indicates that the kidney may adapt to hypoxia by reducing its O2 demand keeping unaltered its tubular function and basal O2 needs.

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.

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).

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.

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.

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.

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.

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the DeltapI(deox.-ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are ;stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (DeltalogP(50)/DeltapH) in solutions, the DeltapI(deox.-ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the DeltapI(deox.-ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A(0) (alpha(2)beta(2)) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5 degrees C and very low concentration of KCl. The variation of the DeltapH(deox.-ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of DeltapI(deox.-ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A(0). In haemoglobin A(0) the DeltapI(deox.-ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The DeltapI(deox.-ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the alpha-chains are decreased by 30% compared with the DeltapI value measured in haemoglobin A(0). When the C-terminus of the beta-chains is altered, as in Hb Nancy (alpha(2)beta(Tyr-145-->Asp) (2)), we observed a 70% decrease in the DeltapI value compared with that measured in haemoglobin A(0). These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1alpha and His-146beta, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem.248, 7039-7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol.138, 649-670]. In other mutant haemoglobins it is demonstrated also that the DeltapI(deox.-ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol.138, 649-670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.

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.

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.

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.

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.

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.