Alprazolam induced conformational change in hemoglobin

Alprazolam (ALP) is a widely prescribed sedative and antidepressant benzodiazepine group of drugs. The wide uses of this drug lead us to investigate its possible interaction with hemoglobin (Hb). Spectrophotometric and spectofluorimetric studies showed strong binding of ALP with Hb. Circular dichroic spectra showed that alpha-helical structure of Hb-subunits has been largely changed. On ALP treatment partial pressure of O(2) is increased in the blood indicating release of O(2) from erythrocytes. Further, the binding of ALP-induced conformational changes in Hb resulting in larger Hb particle size was demonstrated by dynamic light scattering experiment. Thus, the present study unambiguously raises question of danger of random usage of ALP, which binds with and changes the function of Hb.     

Rapid spectrophotometric determination of oxygen consumption using hemoglobin, in vitro: light scatter correction and expanded dynamic range

The method of using absorbance in conjunction with hemoglobin (Hb) to monitor rapid changes in oxygen consumption in vitro was improved by using a non-linear calibration technique and multiwavelength spectroscopy. The O(2) dependence of Hb absorbance was effectively linearized using the current technique (R(2) = 0.990+/-0.002, n = 3), and extended the dynamic range of [O(2)] determinations by 1.6-fold over previous approaches. The association/dissociation rates of O(2) and Hb were evaluated using the current approach and were not significant on the 100-ms time domain. A method was also developed for compensating for large amplitude light scattering changes in turbid media using multiwavelength analysis. Both the nonlinear calibration curve and light scattering corrections were validated in isolated porcine heart mitochondrial preparations.     

Consequences of 6 weeks of strength training on red cell O2 transport and iron status

Effects of endurance training on O2 transport and on iron status are well documented in the literature. Only a few data are available concerning the consequences of strenuous anaerobic muscular exercise on red cell function. This study was performed to test the influence of strength training alone on parameters of red cell O2 transport and iron status. Twelve healthy untrained males participated in a strength-training programme of 2-h sessions four times a week lasting 6 weeks. After 6 weeks a small but significant reduction of haemoglobin (Hb; -5.4 g.l-1) was found (p less than 0.05). Mean red cell volume did not change, but a pronounced decrease of mean cell Hb concentration (from 329.2 g.l-1, SE 2.5 to 309.8 g.l-1, SE 1.2; p less than 0.001) and mean corpuscular Hb (from 29.6 pg, SE 0.4 to 27.7 pg, SE 0.3; p less than 0.01) was observed. Serum ferritin decreased significantly by 35% (p less than 0.01); transferrin, serum iron and iron saturation of transferrin were unaltered. Serum haptoglobin concentration was diminished significantly by 30.5% (p less than 0.01). The reticulocyte count had already increased after 3 weeks of training (p less than 0.05) and remained elevated during the following weeks. Strength training had no significant influence on the O2 partial pressure at which Hb under standard conditions was 50% saturated, red cell 2,3-diphosphoglycerate and ATP concentration as well as on erythrocytic glutamate-oxalacetate transaminase activity. The data demonstrate that mechanical stress of red cells due to the activation of large muscle masses led to increased intravascular haemolysis, accompanied by a slightly elevated erythropoiesis, which had no detectable influence on Hb-O2 affinity. Training caused an initial depletion of body iron stores (prelatent iron deficiency). Although Hb had decreased by the end of the training phase a true "sports anaemia" could not be detected.     

Haemoglobin-induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids

Patients with increased haemolytic haemoglobin (Hb) have 10-20-times greater incidence of cardiovascular mortality. The objective of this study was to evaluate the role of Hb peroxidase activity in LDL oxidation. The role of Hb in lipid peroxidation, H(2)O(2) generation and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed using NaN(3), a peroxidase inhibitor, catalase, a H(2)O(2) decomposing enzyme and human umbilical vein endothelial cells (HUVECs), respectively. Hb induced H(2)O(2) production by reacting with LDL, linoleate and cell membrane lipid extracts. Hb-induced LDL oxidation was inhibited by NaN(3) and catalase. Furthermore, Hb stimulated ICAM-1 and VCAM-1 expression, which was inhibited by the antioxidant, probucol. Thus, the present study suggests that the peroxidase activity of Hb produces atherogenic, oxidized LDL and oxidized polyunsaturated fatty acids (PUFAs) in the cell membrane and reactive oxygen species (ROS) formation mediated Hb-induced ICAM-1 and VCAM-1 expression.     

Deuterium incorporation into Escherichia coli proteins. A neutron-scattering study of DNA-dependent RNA polymerase

Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA-dependent RNA polymerase of Escherichia coli match when RNA polymerase is isolated from cells grown in a medium containing 46% D2O and unlabelled glucose as carbon source. Their contrasts vanish simultaneously in a dialysis buffer containing 65% D2O. An expression was evaluated which allows the calculation of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small-angle scattering results, on which the calculation of the degree of deuteration is based, were confirmed by mass spectrometric measurements.     

O2 release from Hb vesicles evaluated using an artificial,narrow O2-permeable tube: comparison with RBCs and acellular Hbs

A phospholipid vesicle that encapsulates a concentrated hemoglobin (Hb) solution and pyridoxal 5'-phosphate as an allosteric effector [Hb vesicle (HbV) diameter, 250 nm] has been developed to provide an O2 carrying ability to plasma expanders. The O2 release from flowing HbVs was examined using an O2-permeable, fluorinated ethylenepropylene copolymer tube (inner diameter, 28 microm) exposed to a deoxygenated environment. Measurement of O2 release was performed using an apparatus that consisted of an inverted microscope and a scanning-grating spectrophotometer with a photon-count detector, and the rate of O2 release was determined based on the visible absorption spectrum in the Q band of Hb. HbVs and fresh human red blood cells (RBCs) were mixed in various volume ratios at a Hb concentration of 10 g/dl in isotonic saline that contained 5 g/dl albumin, and the suspension was perfused at the centerline flow velocity of 1 mm/s through the narrow tube. The mixtures of acellular Hb solution and RBCs were also tested. Because HbVs were homogeneously dispersed in the albumin solution, increasing the volume of the HbV suspension resulted in a thicker marginal RBC-free layer. Irrespective of the mixing ratio, the rate of O2 release from the HbV/RBC mixtures was similar to that of RBCs alone. On the other hand, the addition of 50 vol% of acellular Hb solution to RBCs significantly enhanced the rate of deoxygenation. This outstanding difference in the rate of O2 release between the HbV suspension and the acellular Hb solution should mainly be due to the difference in the particle size (250 vs. 7 nm) that affects their diffusion for the facilitated O2 transport.     

Erythropoiesis in normal and mutant chick embryos

Hemoglobin D_Davis (Hb D_D), an autosomal codominant in chickens, the α^D-globin chain of Hb M of primitive cells and Hb D of definitive erythrocytes. Erythropoiesis and Hb synthesis was investigated in normal, heterozygous, and homozygous Hb D_D mutant embryos (stages 15–44) and adults. The time of appearance, morphology, relationships to developmental changes, and number of primitive and definitive cells were determined. Primitive hemoglobins between stages 17 and 44 showed four components, P1, P2, E, and M (or M_D), on high-resolution isoelectric focusing gels. Comparison of $\text{P1}\text{P2}$ ratios in the four phenotypes indicated that homozygous Hb D_D embryos had an increased proportion of Hb P2 relative to Hb P1 between stages 17 and 35. This difference coincided with an increase in the number of large primitive cells. In all phenotypes the proportions of primitive hemoglobins decreased after stage 25 and they were not detected after stage 40. Basophilic definitive erythroblasts were present in cell suspensions from all phenotypes between stages 24 and 25. Hb A, the major Hb and Hb D, the minor Hb, of definitive cells of embryos and adults were detected by isoelectric focusing of lysates by stage 29. Definitive cells from late embryos of all phenotypes had higher proportions of Hb D (or Hb D_D) than did red cells from corresponding adult birds. Heterozygous Hb D_D embroys and adults had both Hb D and Hb D_D. Hb D_D comprises about 30% of the total minor Hb rather than 50% expected for heterozygosity at a single locus. In this respect heterozygous Hb D_D chick embryos and adult birds are similar to certain heterozygous α-chain variants in humans. A minor Hb, H, found in lysates of later embryos disappears in lysates of normal chicks 65 days after hatching, but was present in the circulation of homozygous Hb D_D chicks until at least 195 days after hatching. Additionally, several minor Hb components which may be asymmetrical hybrids or derived precursors of Hb A and Hb D (or Hb D_D) were observed. This study provides the precise developmental stages when the switchover of erythroid cell populations and hemoglobins in the chick embryo occurs. This is the first investigation of an α-globin chain mutant which is synthesized during all stages of red cell development and may be a useful animal model for the study of hemoglobinopathies in vertebrates.     

Mechanism of oxidative damage to fish red blood cells by ozone

The present study was conducted to elucidate the adverse effects of ozone exposure on rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs). We evaluated whether hemoglobin (Hb) or Hb-derived free iron could participate in the RBC damage using an in vitro ozone exposure system. Ozone exposure induced hemolysis, formation of methemoglobin, and RBC membrane lipid peroxidation. This RBC damage was not suppressed by the addition of a specific iron chelator (deferoxamine mesilate) to the medium but was suppressed by carbon monoxide (CO) treatment before ozone exposure. Generation of hydrogen peroxide (H2O2) in RBC was observed upon ozone exposure but was significantly suppressed by CO treatment before ozone exposure. Thus the Hb status (i.e., Hb redox condition) and H2O2 generation in RBC should play important roles in mediating RBC damage by ozone exposure. In other words, neither ozone nor its derivative directly attacked from the outside of the cell, but ozone that penetrated through the membrane derived the reactive oxygen species from Hb inside of the cell.     

Properties of a recombinant human hemoglobin with aspartic acid 99(beta), an important intersubunit contact site, substituted by lysine.

Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.     

Environmentally-related changes in red cell levels of ionic modulators of hemoglobin-O2 affinity in rainbow trout, Salmo gairdneri

Hemoglobin content, plasma and red cell levels of chloride and magnesium and molar ion:hemoglobin ratios were examined in trout acclimatized to eight combinations of two treatment levels of temperature (5, 20 degrees C), O2 availability (less than or equal to 30%, greater than or equal to 75% saturation) and photoperiod (16L:8D, 8L:16D). Increases in hemoglobin content were associated with exposure to higher temperature, abbreviated daylength and hypoxia, with hypoxia greater than photoperiod greater than temperature. Under nominal "summer" conditions (20 degrees C, hypoxia, 16L:8D) photoperiodic influence was apparently masked by hypoxic and thermal effects. Temperature was the principal determinant of plasma and cellular chloride levels as well as [Cl:Hb]. O2 availability and photoperiod had little effect. Temperature was also the primary factor influencing magnesium, with hypoxia exerting a lesser influence. Photoperiod effects were negligible. With increased temperature and reduced O2 availability, plasma magnesium increased white cell magnesium levels and [Mg:Hb] declined. These observations suggest that with normal seasonal changes in environmental conditions, temperature-induced increases in the O2 requirements of summer trout are probably accompanied by increases in blood O2-carrying capacity and reductions in hemoglobin-O2 affinity with consequent increases in O2 delivery to tissues.     

Hemoglobin S and some other hemoglobinopathies in Eti-Turks

Frequencies of various hemoglobinopathies were examined in a total of 1,922 individuals of Eti-Turk origin by electrophoretical techniques. Hemoglobin A2 (Hb A2) and hemoglobin F (Hb F) determinations were also performed in 651 and 1,642 cases, respectively. Mean hemoglobin S (Hb S) frequency was found to be 15.3%. Variations among the different age groups were insignificant. Hemoglobin E (Hb E) and beta-thalassemia frequencies wer 0.47 and 1.23%, respectively. Hemoglobin Hacettepe and hemoglobin D were found once. Red cell glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in 6.5% of males.     

Roles of hemoglobin Allostery in hypoxia-induced metabolic alterations in erythrocytes: simulation and its verification by metabolome analysis

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [(13)C]glucose to [(13)C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O(2)-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O(2) from the cells.     

Effect of zeta-globin substitution on the O2-transport properties of Hb S in vitro and in vivo

Hemoglobin zeta(2)beta(2)(S) is generated by substituting embryonic zeta-globin subunits for the normal alpha-globin components of Hb S (alpha(2)beta(2)(S)). This novel hemoglobin has recently been shown to inhibit polymerization of Hb S in vitro and to normalize the pathological phenotype of mouse models of sickle cell disease in vivo. Despite its promise as a therapeutic tool in human disease, however, the basic O(2)-transport properties of Hb zeta(2)beta(2)(S) have not yet been described. Using human hemoglobins purified from complex transgenic-knockout mice, we show that Hb zeta(2)beta(2)(S) exhibits an O(2) affinity as well as a Hill coefficient, Bohr response, and allosteric properties in vitro that are suboptimally suited for physiological O(2) transport in vivo. These data are substantiated by in situ analyses demonstrating an increase in the O(2) affinity of intact erythrocytes from mice that express Hb zeta(2)beta(2)(S). Surprisingly, though, co-expression of Hb zeta(2)beta(2)(S) leads to a substantial improvement in the tissue oxygenation of mice that model sickle cell disease. These analyses suggest that, in the context of sickle cell disease, the beneficial antisickling effects of Hb zeta(2)beta(2)(S) outweigh its O(2)-transport liabilities. The potential structural bases for the antisickling properties of Hb zeta(2)beta(2)(S) are discussed in the context of these new observations.     

Pericardial delivery of omega-3 fatty acid: a novel approach to reducing myocardial infarct sizes and arrhythmias

O2-carrying fluids based on hemoglobin (Hb) are in various stages of clinical trials to determine their suitability as O2-carrying plasma expanders. Polymerized Hb solutions are characterized by their vasoactivity, low O2 affinity, oncotic effect, prolonged shelf life, and stability. Physiological responses to facilitated O2 transport after exchange transfusion with polymerized bovine Hb (PBH) were studied in the hamster window chamber model during acute moderate anemia to determine how PBH affects microvascular perfusion and tissue oxygenation. The anemic state [29% hematocrit (Hct)] was induced by hemodilution with a plasma expander (70 kDa dextran). After hemodilution, animals were randomly assigned to different exchange transfusion groups. Study groups were based on the concentration of PBH used, namely: PBH at 13 g Hb/dl [PBH13], PBH diluted to 8 (PBH8) or 4 (PBH4) g Hb/dl in albumin solution at matching colloidal osmotic pressure (COP), and no PBH (only albumin solution) at matching COP (PBH0). Measurement of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O2 levels was performed at 18% Hct. Restitution of O2-carrying capacity with PBH13 increased arterial pressure and triggered vasoconstriction, low perfusion, and high peripheral resistance. PBH4 and PBH0 exhibited lower arterial pressures compared with PBH13. Exchange transfused animals with PBH8 and PBH4 better maintained perfusion and functional capillary density than PBH13. Blood gas parameters and acid-base balance were recovered proportional to microvascular perfusion. Arterial O2 tensions were improved with PBH4 and PBH8 by preventing O2 precapillary release and increasing O2 reserve. Further studies to establish PBH optimal dosage, efficacy, safety, and its effect on outcome are indicated before Hb-based O2-carrying blood substitutes are implemented in routine practice.     

Interactions of hemoglobin with hydrogen peroxide alters thiol levels and course of endothelial cell death

We investigated cellular injury and death induced by ultrapure human Hb (HbA(0)) and its diaspirin cross-linked derivative DBBF-Hb in normal and glutathione (GSH)-depleted bovine aortic endothelial cells subjected to hydrogen peroxide (H(2)O(2)). HbA(0) underwent extensive degradation and heme loss, whereas DBBF-Hb persisted longer in its ferryl (Fe(4+)) form. The formation of ferryl HbA(0) or ferryl DBBF-Hb was associated with a significant decrease in endothelial cell GSH compared with the addition of H(2)O(2) or Hbs alone. This effect was inhibited by catalase, but not by superoxide dismutase or deferoxamine mesylate. The presence of HbA(0) and DBBF-Hb reduced H(2)O(2)-induced apoptosis, as measured by cell morphology, annexin V binding assay, and caspase inhibition, consistent with the ability to consume H(2)O(2) in an enzyme-like fashion. However, the pattern of cell death and injury produced by HbA(0) and DBBF-Hb appeared to be distinctly different among proteins as well as among cells with and without GSH. These findings may have important implications for the use of cell-free Hb as oxygen therapeutics in patients with coexisting pathologies who may lack antioxidant protective mechanisms.     

Solvent isotope effect on macromolecular dynamics in <Emphasis Type="Italic">E. coli</Emphasis>

Elastic incoherent neutron scattering was used to explore solvent isotope effects on average macromolecular dynamics in vivo. Measurements were performed on living E. coli bacteria containing H(2)O and D(2)O, respectively, close to physiological conditions of temperature. Global macromolecular flexibility, expressed as mean square fluctuation (MSF) values, and structural resilience in a free energy potential, expressed as a mean effective force constant, [Symbol: see text]k'[Symbol: see text], were extracted in the two solvent conditions. They referred to the average contribution of all macromolecules inside the cell, mostly dominated by the internal motions of the protein fraction. Flexibility and resilience were both found to be smaller in D(2)O than in H(2)O. A difference was expected because the driving forces behind macromolecular stabilization and dynamics are different in H(2)O and D(2)O. In D(2)O, the hydrophobic effect is known to be stronger than in H(2)O: it favours the burial of non-polar surfaces as well as their van der Waals' packing in the macromolecule cores. This may lead to the observed smaller MSF values. In contrast, in H(2)O, macromolecules would present more water-exposed surfaces, which would give rise to larger MSF values, in particular at the macromolecular surface. The smaller [Symbol: see text]k'[Symbol: see text] value suggested a larger entropy content in the D(2)O case due to increased sampling of macromolecular conformational substates.     

Optimal hemoglobin concentration and high altitude: a theoretical approach for Andean men at rest.

The beneficial role of erythrocytosis for O2 transport has been questioned by evidence from bloodletting and hemodilution research as well as by studies suggesting the existence of an "optimal" hematocrit (Hct) or hemoglobin concentration ([Hb]) value. To assess to what extent erythrocytosis is beneficial in Andean men at high altitude, we examined and discussed optimal [Hb] using a mathematical approach by modeling the mixed (mean) venous Po2 (Pv(O2)) and arterial O2 content, considering for both the relation between [Hb] and arterial Po2. Relations of [Hb] to other physiological variables such as cardiac output and convective arterial O2 transport were also discussed, revealing the importance of Pv(O2) in this model. Our theoretical analysis suggests that increasing [Hb] allows increase and maintenance of Pv(O2) with only moderate declines in arterial Po2 as a consequence of moderate increases in altitude, reaching its maximum at the optimal [Hb] of 14.7 g/dl. Our analysis also shows that [Hb] corresponding to high arterial O2 content and O2 transport values is apparently not quite advantageous for improvement of oxygenation. Furthermore, chronic mountain sickness is discussed as an insightful example of the effects of excessive erythrocytosis at high altitude.     

Nuclear magnetic resonance and oxygen affinity study of cesium binding in human erythrocytes.

We investigated the interaction of the cesium ion (Cs(+)) with the anionic intracellular components of human red blood cells (RBCs); the components studied included 2,3-bisphosphoglycerate (BPG), ADP, ATP, inorganic phosphate (P(i)), carbonmonoxy hemoglobin (COHb), and RBC membranes. We used spin-lattice (T(1)) and spin-spin (T(2)) (133)Cs NMR relaxation measurements to probe Cs(+) binding, and we found that Cs(+) bound more strongly to binding sites in BPG and in RBC membranes than in any other intracellular component in RBCs at physiologic concentrations. By using James-Noggle plots, we obtained Cs(+) binding constants per binding site in BPG (66 +/- 8 M(-1)), ADP (19 +/- 1 M(-1)), ATP (25 +/- 3 M(-1)), and RBC membranes (55 +/- 2 M(-1)) from the observed T(1) values. We also studied the effect of Cs(+) on the oxygen (O(2)) affinity of purified Hb and of Hb in intact RBCs in the absence and in the presence of BPG. In the absence of BPG, the O(2) affinity of Hb decreased upon addition of Cs(+). However, in the presence of BPG, the O(2) affinity of Hb increased upon addition of Cs(+). The O(2) affinity of Cs(+)-loaded human RBCs was larger than that of Cs(+)-free cells at the same BPG level. (31)P NMR studies on the pH dependence of the interaction between BPG and Hb indicated that the presence of Cs(+) resulted in a smaller fraction of BPG available to bind to the cleft of deoxyHb. Our NMR and O(2) affinity data indicate that a strong binding site for Cs(+) in human RBCs is BPG. A partial mechanism for Cs(+) toxicity might arise from competition between Cs(+) and deoxyHb for BPG, thereby increasing oxygenation of Hb in RBCs, and thus decreasing the ability of RBCs to give up oxygen in tissues. The presence of Cs(+) at 12.5 mM in intact human RBCs containing BPG at normal concentrations did not, however, alter significantly the O(2) affinity of Hb, thus ruling out the possibility of Cs(+)-BPG interactions accounting for Cs(+) toxicity in this cell type.     

The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.     

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O(2) carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs.