Fetal hemoglobin distributions among adult baboon and macaque species

Heritable variation in fetal hemoglobin (Hb F) in erythrocytes of the adult human has been shown to occur at more than one genetic locus. Heritable variation has also been reported in adult baboons. Nonhuman primates thus may serve as useful models for understanding how Hb F is regulated in the human. In the study reported here we identified Hb F in hemolysates from 27 of 32 rhesus macaques, from 32 of 32 baboons, and from none of 35 cynomoglus macaques. Hb F as a percentage of total hemoglobin occurred as a normally distributed variable among rhesus macaques but among baboons the distribution was both skewed and kurtotic. Such difference could be either a consequence of nonrandom sampling of the gene pool in one of the species, or a consequence of species evolution. A technique of single cell hemoglobin electrophoresis was applied to erythrocytes from three adult pig-tailed macaques. This demonstrated that erythrocytes which contain Hb F (F-cells) also customarily contain Hb A and that the proportions of these two hemoglobins varies substantially among the F-cells, as we previously noted for human F-cells. We conclude that the macaques could serve as useful models for understanding Hb F regulation in the human.     

Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electrosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The ∼90 fl injection volume for individual cells is 7×10⁴ times lower than those previously reported. Adult (normal and elevated A₁), sickle (heterozygous), and fetal erythrocytes were analyzed, with the amounts of hemoglobins A₀, A_1c, S and F determined. The pH gradient for cIEF was linear (r² = 0.9984), which allowed tentative identification of Hb F_ac. Variants differing by as little as 0.025 pI units were resolved.     

The metamorphic switch in hemoglobin phenotype ofXenopus laevis involves erythroid cell replacement

Summary To elucidate the cellular basis of hemoglobin transition inXenopus laevis the distribution of larval and adult hemoglobins was analyzed by indirect immunofluorescence in the circulating erythrocytes during metamorphosis. In addition, the morphological characteristics as well as the capacity for synthesis of DNA and hemoglobin in the erythrocytes were followed during the same developmental period. Our quantitative analysis on the distribution of larval and adult hemoglobins suggests that they are localized in different cells. Hemoglobin transition, therefore, most likely reflects replacement of the larval erythrocyte population by new cells which are committed to adult globin synthesis. Since hemoglobin transition is not accompanied by an increase in the abundance of immature erythroid cells with active DNA synthesis, we assume that the presumptive adult erythroid cells are released into circulation at a relatively advanced stage of maturation. The decline in the synthesis of DNA and larval hemoglobin further indicates that cessation of cell renewal in the larval erythrocyte population may represent a decisive step in hemoglobin transition.     

Genetic hemoglobin abnormalities in 2363 Cuban newborns

Summary A total of 2363 Cuban newborns were screened for genetic hemoglobin abnormalities; 2187 (92.56%) had a normal electrophoretic pattern. Of the 176 samples with abnormal electrophoretic patterns, 102 (4.32%) had hemoglobins A, F plus Bart's; 54 (2.29%) had hemoglobins A, F and S; 3 (0.13%) had hemoglobins A, F, S plus Bart's; 14(0.59%) had hemoglobins A, F and C; 1 (0.04%) had hemoglobins A, F, C and Bart's. The frequency of Hb Bart's was 4.46% in AA phenotype, 5.25% in AS, and 6.67% in AC. Two newborns were found to have rare variants. A close correlation was found between the observed and expected phenotypes, which indicates the accuracy of the diagnostic methods used. The results of all hemoglobin abnormalities were entered on the infants' hospital records. In addition, these families received genetic counseling.     

The acetylation state of human fetal hemoglobin modulates the strength of its subunit interactions: long-range effects and implications for histone interactions in the nucleosome

The source of the 70-fold increased tetramer strength of liganded fetal hemoglobin relative to that of adult hemoglobin between pH 6.0 and 7.5 reported earlier [Dumoulin et al. (1997) J. Biol. Chem. 272, 31326] has been identified as the N-terminal Gly residue of the gamma-chain, which is replaced by Val in adult hemoglobin. This was revealed by extending the study of the pH dependence of the tetramer-dimer equilibrium of these hemoglobins into the alkaline range as far as pH 9. From pH 7.5 to 9.0, the 70-fold difference in the association equilibrium constant between hemoglobins F and A lessened progressively. This behavior was attributed to the difference in the pK(a) 8.1 of Gly-1(gamma) compared to the pK(a) 7.1 value of Val-1(beta) of hemoglobins F and A, respectively. Evidence for this conclusion was obtained by demonstrating that natural hemoglobin F(1), which is specifically acetylated at Gly-1(gamma) and hence unable to be protonated, behaves like HbA and not HbF in its tetramer-dimer association properties over the pH range studied. An increased degree of protonation of the gamma-chain N-terminus of hemoglobin F from pH 9.0 to 8.0 is therefore suggested as responsible for its increased tetramer strength representing an example of transmission of a signal from its positively charged N-terminal tail to the distant subunit allosteric interface where the equilibrium constant is measured. An analogy is made between the effects of acetylation of the fetal hemoglobin tetramer on the strength of its subunit interactions and acetylation of some internal Lys residues within the N-terminal segments of the histone octamer around which DNA is wrapped in the nucleosome.     

Identification of eleven human hemoglobin variants by high-performance liquid chromatography: Additional data on functional properties and clinical expression

Eleven abnormal hemoglobins were detected in the course of cord blood screening or in the evaluation of evident hematological problems in individual cases. Identification of the variant in each case was done by high-performance liquid chromatography (HPLC); HPLC provides a rapid, sensitive means for the examination of abnormal hemoglobins. Some of the 11 variants that were identified have been described repeatedly and are included to provide information on the HPLC behavior of tryptic peptides. Others are much rarer. Additional information is provided about the hematological and clinical expression as well as ethnic and geographical distribution of the abnormal hemoglobin.This investigation was supported in part by Grants HL-02558 and HL-15162 from the National Institutes of Health, U.S. Public Health Service.     

Phenoxazinone synthesis by human hemoglobin.

We found that 2-amino-5-methylphenol was converted to the dihydrophenoxazinone with a reddish brown color by purified human hemoglobin, lysates of human erythrocytes, and human erythrocytes. The reddish brown compound was identified as 2-amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazin-3-one by the measurement of NMR spectra, IR spectra, EI mass spectra, and absorption spectra. The changes in this phenoxazinone were studied under various conditions after mixing 2-amino-5-methylphenol with purified oxy- or methemoglobin, or with human erythrocytes. The production of 2-amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazine-3-one from 2-amino-5-methylphenol was found to be tightly coupled with the oxidation of ferrous hemoglobin and reduction of ferric hemoglobin under aerobic conditions. By studying the production rates of the dihydrophenoxazinone and the oxido-reduction rates of ferrous and ferric hemoglobins during the reactions of ferrous or ferric hemoglobin with 2-amino-5-methylphenol under aerobic and anaerobic conditions, the reaction mechanism was extensively proposed.     

Subunit dissociation in fish hemoglobins.

The tetramer-dimer dissociation equilibria (K 4,2) of several fish hemoglobins have been examined by sedimentation velocity measurements with a scanner-computer system for the ultracentrifuge and by flash photolysis measurements using rapid kinetic methods. Samples studied in detail included hemoglobins from a marine teleost, Brevoortia tyrannus (common name, menhaden); a fresh water teleost, Cyprinus carpio, (common name, carp); and an elasmobranch Prionace glauca (common name, blue shark). For all three species in the CO form at pH 7, in 0.1 M phosphate buffer, sedimentation coefficients of 4.3 S (typical of tetrameric hemoglobin) are observed in the micromolar concentration range. In contrast, mammalian hemoglobins dissociate appreciably to dimers under these conditions. The inability to detect dissociation in three fish hemoglobins at the lowest concentrations examined indicates that K 4,2 must have a value of 10(-8) M or less. In flash photolysis experiments on very dilute solutions in long path length cells, two kinetic components were detected with their proportions varying as expected for an equilibrium between tetramers (the slower component) and dimers (the faster component); values of K 4,2 for the three fish hemoglobins in the range 10(-9) to 10(-8) M were calculated from these data. Thus, the values of K 4,2 for liganded forms of the fish hemoglobins appear to be midway between the value for liganded human hemoglobin (K 4,2 approximately 10(-6) M) and unliganded human hemoglobin (K 4,2 approximately 10(-12) M). This conclusion is supported by measurements on solutions containing guanidine hydrochloride to enhance the degree of dissociation. All three fish hemoglobins are appreciably dissociated at guanidine concentrations of about 0.8 M, which is roughly midway between the guanidine concentrations needed to cause comparable dissociation of liganded human hemoglobin (about 0.4 M) and unliganded human hemoglobin (about 1.6 M). Kinetic measurements on solutions containing guanidine hydrochloride indicated that there are changes in both the absolute rates and the proportions of the fast and slow components, which along with other factors complicated the analysis of the data in terms of dissociation constants. Measurements were also made in solutions containing urea to promote dissociation, but with this agent very high concentrations (about 6 M) were required to give measureable dissociation and the fish hemoglobins were unstable under these conditions, with appreciable loss of absorbance spectra in both the sedimentation and kinetic experiments.     

Diagnosis of Hemoglobinopathies by High-Performance Liquid Chromatography

This report describes a unique cation exchange high-performance liquidchromatography capable of separating more than 40 frequently encountered human hemoglobins and variants within 12 min. Some of these variants are unresolvable by the conventional electrophoretic methods and would thus lead to an incorrect diagnosis of hemoglobinpathy. The method provides high sensitivity, superior resolution and accurate quantitation of hemoglobin concentrations. It can also be fully automated thus make it an ideal methodology for the diagnosis of hemoglobin disorders in a routine clinical laboratory.     

Hemoglobin aggregation in oxygenated sickle cells studies by carbon-13 rotating frame spin-lattice relaxation in the presence of an off-resonance radiofrequency field

Evidence is presented which shows that hemoglobin S in sickle cells has a tendency to aggregate even in the oxygenated state. The basis for that conclusion is derived from ¹³C-nmr rotating-frame spin–lattice relaxation studies in the presence of an off-resonance radiofrequency field in which the carbonyl resonances of hemoglobins in erythrocytes are examined. The experiments indicate that the rotational correlation time of hemoglobin S in oxygenated sickle cells at 38°C is 130 nsec compared to a value of 95 nsec for hemoglobin A in normal erythrocytes at the same temperature and the same mean cell hemoglobin content.     

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

Plasma 125I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the 125I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies.     

Separation of asymmetrical hybrid containing hemoglobin F by anaerobic anion-exchange high-performance liquid chromatography

Asymmetrical hybrid hemoglobins formed from mixtures of oxyhemoglobins S and F and A and F were separated by high-performance liquid chromatography on a 4.6 X 250 mm wide-pore polyethyleneimine-silica gel column under anaerobic conditions. The resulting HPLC chromatogram showed three peaks, with the middle peak representing the hybrid hemoglobin. The areas of these three peaks were quantified and the amount of hybrids formed was less than that predicted theoretically. We found that the deviation was due to the equilibrium constant of the FS hybrid hemoglobin differing from that of the parent hemoglobins. In this report, we introduce the anaerobic recycle ion-exchange HPLC method to determine the rate of dissociation of AS and FS hybrid hemoglobins at constant pH buffer conditions. The results obtained by this method demonstrate that FS hybrid hemoglobin is more unstable than AS hybrid hemoglobin. The free energy of association for asymmetrical hybrids containing hemoglobin F is approximately 0.6 Kcal/mol greater than that of the symmetrical parent hemoglobins.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

ERYTHROPOIESIS DURING AMPHIBIAN METAMORPHOSIS : III. Immunochemical Detection of Tadpole and Frog Hemoglobins (Rana catesbeiana) in Single Erythrocytes

Rabbit antibodies specific for the major tadpole and frog hemoglobin components of R. catesbeiana were used for the detection of the two hemoglobins inside single cells. The antisera, after fractionation by ammonium sulfate precipitation and diethylaminoethyl (DEAE)-cellulose chromatography, were conjugated with fluorescein isothiocyanate for the antifrog hemoglobin serum and tetramethylrhodamine isothiocyanate for the antitadpole hemoglobin serum. The conjugated fractions, refractionated by stepwise elution from a DEAE-cellulose column, were used for the fluorescent staining of blood smears, liver tissue imprints, and smears of liver cell suspensions. Both simultaneous and sequential staining with the two fluorescent preparations indicated that larval and adult hemoglobins were not present within the same erythrocyte during metamorphosis. In other experiments, erythroid cells from animals in metamorphosis were spread on agar containing specific antiserum. Precipitates were formed around the cells which contain the particular hemoglobin. The percentages of cells containing either tadpole or frog hemoglobin were estimated within the experimental error of the method. The data showed that the two hemoglobins are in different cells. It is concluded that the hemoglobin change observed during the metamorphosis of R. catesbeiana is due to the appearance of a new population of erythroid cells containing exclusively frog hemoglobin.     

Differential control of the synthesis of two hemoglobin beta chains in normal mice

A fetal-to-adult switch in the proportion of the mouse minor hemoglobin is described. Although mice have no fetal hemoglobin per se, the timing of this switch in the mouse suggests that the mechanism of its control may directly parallel that of the human switch from fetal to adult hemoglobin expression. The mouse minor hemoglobin is expressed only in strains with the "diffuse" allele for the beta chain complex locus. Fetal liver cells of these mice synthesize a much greater proportion of the betaminor globin chain that do adult hematopoietic cells. Consequently, circulating fetal erythrocytes carry a high level of the minor hemoglobin containing it. By the time of birth, a lowered proportion of betaminor is synthesized in the liver. This low proportion continues to be expressed during early erythroid maturation in the adult. The fetal-to-adult switch is the first indication that in normal mice, the two beta chain loci can be expressed noncoordinately. The similarity between the patterns of the decline of the minor hemoglobin in mice and of the disappearance of fetal hemoglobin in humans suggests that the minor hemoglobin in the "diffuse" mouse may function to some degree as a fetal hemoglobin in the period between the disappearance of the embryonic hemoglobins and the time of birth.     

Phagocytosis of erythrocytes by the proximal tubule of the rat kidney

Summary Morphological examination of kidney biopsies from patients with glomerulonephritis and hematuria has revealed the presence of erythrocytes within epithelial cells of the proximal tubule. This observation suggested that the proximal tubule might be capable of phagocytizing morphologically intact erythrocytes. To examine this possibility small quantities of heparinized autologous blood were injected into surface convolutions of proximal tubules of the rat kidney using standard micropuncture techniques. At time intervals ranging from 10 min to 120 h after injection, the kidneys were preserved for light and transmission electron microscopy by drip-fixation with a half-strength Karnovsky's glutaraldehyde-formaldehyde fixative.During the initial 6 h there was a flattening of the brush border and accumulation of electron-dense material representing hemoglobin in apical vacuoles and in lysosome-like structures. From 6 to 15 h after micropuncture, there was progressive loss of the brush border and the simultaneous formation of pseudopodia-like evaginations that extended from the apical plasma membrane and surrounded the individual erythrocytes. By 18 and 24 h, erythrocytes were observed in the proximal tubule cells. At later time intervals, edema, lymphocytic infiltration, and fibrosis were observed in the interstitium. In addition, crystalline structures were present in the lumen and the cells of both proximal and distal tubules. These findings suggest that in addition to their well-established ability to pinocytize hemoglobin and other proteins, the cells of the proximal tubule are capable of phagocytizing morphologically intact autologous erythrocytes. It is possible that phagocytosis by the proximal tubule cells may play a role in the disposal of erythrocytes from the tubular fluid in hematuric conditions.     

Intraerythrocytic organic phosphates of fetal and adult seaperch (Embiotoca lateralis): Their role in maternal-fetal oxygen transport

Summary The viviparous seaperch,Embiotoca lateralis, has unique fetal and adult hemoglobins. Stripped fetal hemoglobin has a higher oxygen affinity than stripped adult hemoglobin at pH 6.5–7.1. The oxygen affinities of both adult and fetal hemoglobins are lowered allosterically by ATP at pH 7.1. Both fetal and adult seaperch erythrocytes include approximately 82% ATP and 18% GTP of the total nucleotide triphosphates (NTP) with a trace of AMP. No 2,3-diphosphoglycerate or inositol polyphosphate was detected. Mid- and late-gestation erythrocytes contain less NTP/mole hemoglobin tetramer than do adult cells. The effective NTP concentration in adult cells is higher than that of the fetal erythrocytes even when the intracellular concentration of Mg²⁺, which complexes with NTP, is accounted for. The difference in adult and fetal intraerythrocytic NTP concentration should enhance transfer of oxygen from maternal to fetal blood. Thus, the teleostEmbiotoca lateralis may employ a dual mechanism in maternal-fetal oxygen transfer. A difference in fetal and maternal hemoglobin structure and oxygen affinities is enhanced by a difference in their respective intraerythrocytic organic phosphate concentrations.     

Detection of hemoglobin variants in erythrocytes by flow cytometry

BACKGROUND: With the emergence of fetal hemoglobin (Hb F)stimulating agents as potential treatments for sickle-cell disease and thalassemias, procedures to monitor the effect of these agents on Hb F levels in individuals will be needed. We developed a rapid procedure that detects fetal hemoglobin in erythrocytes (F cells) using a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against Hb F. METHODS: Ten microliters of washed blood was fixed in formaldehyde and glutaraldehyde, then permeabilized in a Triton X-100/PBS solution containing a FITC-labeled monoclonal antibody to Hb F. The blood was analyzed by flow cytometry to determine the percentage of F cells. RESULTS: Nearly 200 Hb F-containing samples were analyzed by this protocol and demonstrated good correlation to percent Hb F results determined by high pressure liquid chromatography (HPLC). In addition, a number of samples were fixed and permeabilized using this method as well as a previously-described method that uses dimethyl 3,3'dithiobispropionimadate (DTBP) as a fixative as well as a different anti-Hb F monoclonal. Good correlation (r = 0.96, r2 = 0.93, P<0.001) was observed between the two protocols. CONCLUSIONS: This procedure is easy, reproducible, and gives accurate F cell results. It can be used to measure a wide range of F cell percentages and may also be used to dual-stain Hb F along with other hemoglobin variants and erythrocyte surface antigens.     

Factors in the evolution of hemoglobin function.

The packaging of vertebrate blood hemoglobins within cells places subtle constraints on hemoglobin evolution. Since the concentration of hemoglobin is near the solubility limit a selective advantage should exist for a noncomplementary external topology of amino acid residues. Further, any change in charge on the protein should alter ion distribution across the cell membrane and so modify ion-sensitive oxygen transport. An efficient hemoglobin must not only combine readily with oxygen at prevailing environmental oxygen pressures, but must also release it at metabolically appropriate pressures. These adaptations frequently employ different strategies to achieve the same objective in different animals. Some hemoglobins have evolved special properties unrelated to the transport of oxygen to metabolizing tissues. Thus many teleost fish have hemoglobins that discharge much of their oxygen at low pH even at high oxygen pressures. This property appears to aid in filling the swim bladder with oxygen. The hemoglobins of elasmobranchs have evoked a unique resistance to urea as a consequence of the high urea content of their blood. Sometimes the functional adaptations of hemoglobins are achieved by multiple hemoglobins in the same cells. Often, however, different red cell populations with functionally unique hemoglobins arise sequentially during ontogeny.