Evidence for Duplication of the Haemoglobin γ Chain Locus in <Emphasis Type="Italic">Macaca nemestrina</Emphasis>

FOETAL haemoglobins are synthesized by several and perhaps all, of the Anthropoidea during intrauterine development; little is known of the primary structures of their γ chains, but they are presumed to bear close structural resemblances to human γ chains. Until recently, the synthesis of human γ chains was thought to be controlled by a single structural locus; Schroeder et al.¹ showed that at least two and possibly four, γ-chain loci are active in human foetuses. So far theirs has been the only report indicating that there are two or more γ-chain loci in any mammal. We have evidence for the existence of duplicate haemoglobin γ-chain loci in Macaca nemestrina (pig-tailed macaques).     

Hemogloblin α-gene duplication in macaques: Individual Macaca nemestrina with three structurally different α chains

The diversity of hemoglobin phenotypes observed among Malaysian Macaca nemestrina (pig-tailed macaques) has been attributed, in part, to the presence of duplicated α-chain loci in some members of this species. To date, evidence in support of this view has been indirect, consisting of variation in proportions of α^I (Asp₇₁, Gln₇₈) and α^II (Asp₇₁, His₇₈) chains among presumptive heterozygotes. However, the discovery that erythrocytes from some M. nemestrina contain α^I and α^II chains in company with a third, or α^III, chain (Gly₇₁, Gln₇₈) provides direct evidence of duplicated α-chain loci.     

Characterization by polyacrylamide gel electrophoresis of hemoglobins in rabbit embryo, fetus, and adult

The hemoglobin species of rabbit embryo, fetus, and adult were characterized by quantitative polyacrylamide gel electrophoresis (PAGE). Special statistical methods, including joint confidence envelopes for the slope (K_R) and intercept (Y₀) of the Ferguson plot and analysis of covariance were utilized to identify the hemoglobin species. Five embryonic rabbit hemoglobins could be distinguished. Their relative proportions varied with developmental stage. No specific fetal hemoglobin was detected. The two previously known adult hemoglobins were characterized, prepared by isoclectric focusing on polyacrylamide gel (IFPA) and shown to differ by two amino acid substitutions in the β-chain.A general strategy for testing molecular identity by PAGE is outlined.     

Glycosylated minor components of human fetal hemoglobin. Chromatographic separation, identification, and functional characterization

Human adult red cell lysate contains glycosylated minor hemoglobins AIa1, AIa2, AIb, and AIc. Similar minor hemoglobins, designated FIa1, FIa2, Fib, and FIc, have been separated by a Biorex 70 column chromatographic procedure from red cell lysates of newborn children and from an adult homozygote for hereditary persistence of fetal Hb. The minor Hb components were characterized by analyzing for carbohydrate and phosphate contents, by oxygen equilibrium analysis, and by comparing the chromatographic elution profiles of naturally occurring and in vitro synthesized minor components. The results indicate that Hb FIa1, Hb FIa2, and Hb FIc have been formed by the modification of gamma chains of Hb F by reacting with fructose-1,6-P2, glucose-6-P, and glucose, respectively. Hb FIb is a glycoprotein; the mechanism of its formation is unclear. Hb FIa1 and Hb FIa2 had significantly lower oxygen affinities and n values than the other minor components and the major Hb F0. Moreover, 2,3-diphosphoglycerate did not influence the oxygenation of the minor or the major fetal Hb components. Incubations of Hb F with [14C]hexoses and subsequent chromatographic separation of hemoglobins and their globin chains confirm the previous findings that the binding of carbohydrate to Hb involves both specific and nonspecific reactions.     

Fetal hemoglobin in newborn baboons, Papio cynocephalus

Hemoglobins from a newborn baboon, Papio cynocephalus, and from a 15-day-old baboon of the same species were examined for electrophoretic properties of polypeptide chains and were tested for alkali resistance. Hemoglobin of newborn P. cynocephalus appears to be analogous to human fetal hemoglobin. Hemoglobin of newborn P. cynocephalus is composed of α-like and γ-like chains and hemoglobin of adult P. cynocephalus is composed of α-like and β-like chains.     

Evolutionary trends in the hemoglobins of murine animals

The evolutionary origin of murine line based on a phylogenetic tree made on sequence data of ∞-and β-hemoglobin chains, followed by the diversity spectrum of hemoglobin genes in two wild species of murine rodents:Rattus rattus rufescens (house rat) andBandicota indica (bandicoot rat) has been reported. Each house rat contains six hemoglobin types involving two ∞-and three β-chains, which suggests a probable gene duplication at the oc chain locus and a gene triplication at the β-chain locus. Each bandicoot rat contains one ∞-and two β-chains suggesting a probable gene duplication at the β-chain locus. Peptide pattern analysis of the polypeptide chains of these murine hemoglobins further indicates that intraspecies differences among duplicated chains of the same kind are less than interspecies differences among corresponding ∞-and β-chains.     

Comparative solubilities and electrophoresis of microtine hemoglobins.

1. The electrophoretic mobilities of the hemoglobins of 7 taxa of microtines were compared. Microtus oeconomus, M. pennsylvanicus pullatus and M. xanthognatus showed identical 2-band patterns on electrophoresis of their hemoglobins while M. pennsylvanicus tananaensis showed only a single hemoglobin corresponding to the major band of the others. Dicrostonyx rubricatus and D. stevensoni exhibited identical patterns different from the Microtus species. Lemmus sibiricus had a slow hemoglobin component with mobility slightly different from the slow ones of the Microtus species while the fast component appeared the same. 2. Electrophoresis of individual globin chains from hemolysates, purified hemoglobins, and isolated chains indicated a large degree of similarity between the species studied, although there were significant differences in hemoglobin patterns. 3. The minor hemoglobin band in Microtus seems to be the result of a second alpha chain locus as determined from the hemoglobins from hybrids of two subspecies. 4. Salting-out studies indicated differences between hemoglobins that were not detectable by electrophoresis of either whole hemoglobins or isolated chains. 5. M. xanthognathus hemolysate was considerably less soluble than those of M. oeconomus and M. pennsylvanicus pullatus which had essentially the same solubility. 6. The major hemoglobin components of M. pennsylvanicus pullatus and M. xanthognathus were considerably less soluble than either the corresponding unfractionated hemolysates or purified minor components.     

Hemoglobins of baboons, Papio cynocephalus, P. gelada and P. hamadryas

Major hemoglobins of adult Papio cynocephalus, P. gelada, and P. hamadryas and of newborn P. cynocephalus were purified; globins were prepared; and α and β or α and γ chains were separated. The amino acid compositions and aminoterminal groups were determined. These were compared with analogous data for human hemoglobins, other baboon hemoglobins and macaque hemoglobins.     

Oxygen affinities of maternal and fetal hemoglobins of the viviparous seaperch,Embiotoca lateralis

Summary The striped seaperch,Embiotoca lateralis, is a viviparous teleost. The hemoglobins of adult and fetal seaperch are both tetrameric proteins which in their native state appear to be indistinguishable from one another by electrophoresis. However, differences in the subunit structure of maternal versus fetal seaperch hemoglobins can be detected by electrophoresis in urea with a reducing agent, amino acid analyses and peptide maps of the respective proteins. Furthermore, stripped adult and fetal hemoglobins have different oxygen binding affinities at all pH's tested between pH 6.8 and 8.0. Mid-gestation fetal hemoglobin has a higher oxygen affinity than late-gestation fetal hemoglobin which in turn has a higher affinity than that of the adult hemoglobin. All three stripped hemoglobins show a similar Bohr effect (=–0.9). These data suggest that a difference in oxygen affinities exists in vivo between the adult and fetal blood of the seaperchEmbiotoca lateralis and that it can be explained in part by the presence of a structurally unique fetal hemoglobin. This report is the first to provide evidence for a mechanism of maternal-fetal oxygen transfer in a teleost fish.Abbreviations A adult - LF late-gestation fetal - MF mid-gestation fetal (hemoglobins)     

Direct analysis of α- and β-chains of hemoglobins from mammalian blood samples by nanoESI mass spectrometry during in-capillary proteolytic digestion

α- and β-chains of hemoglobins derived from several species were analyzed directly from diluted blood samples by simultaneous in-capillary proteolytic digestion and nanoESI MS and MS/MS analysis. Starting from fresh or frozen and thawed blood samples, sequence coverages of >80% were usually obtained. Only 2 h after resuspension of a dried blood spot, human origin could be demonstrated from data obtained by in-capillary tryptic digestion, nanoESI mass spectrometric analysis, and data base search. A fast and facile differentiation of closely related species by hemoglobin-derived proteolytic “marker peptides” was demonstrated for Asian (Elephas maximus) and African elephants (Loxodonta africana). Finally, amino acid sequences deduced from collision-induced dissociation experiments during in-capillary proteolytic digestion of the corresponding blood samples allowed de novo sequencing of previously unknown sequences of hemoglobin chains of the Patagonian cavy (Dolichotum patagona) and the Persian gazelle (Gazella subgutturosa subgutturosa). 100% of the α-chain sequences and more than 85% of the β-chain sequences were covered for both the species. Additionally, sequence data derived from tandem MS experiments obtained with the Q-Tof analyzer were confirmed by high resolution Fourier-transform ion cyclotron resonance mass spectrometric experiments. Accurate protein mass determination of the intact hemoglobin chains directly from the corresponding blood samples by use of a Fourier-transform ion cyclotron resonance mass spectrometer corroborated the deduced sequences of the respective α-chains. The present study demonstrates that in-capillary digestion allows fast characterization and/or sequencing of hemoglobin chains directly from blood samples.     

Preliminary survey of carnivore hemoglobin compositions

Summary The amino acid differences among 12 chains and 10 chains of carnivore hemoglobins are given. These hemoglobins conform to the substitution rate found for hemoglobins in general. A table compares the differences among known-chain sequences when deduced from tryptic peptide compositions and when taken from actual sequences. Among the carnivore hemoglobins studies, tryptic peptide compositions are about 14 % low in giving the number of sequence differences.     

Purification of three γ-chains with different molecular weights from normal human plasma fibrinogen

Three forms of the normal human plasma fibrinogen γ-chain which differ in molecular weight have been purified. Plasma fibrinogen was separated by ion exchange chromatography on DEAE-Sephacel into three populations of molecules, each with a unique γ-chain composition. Following reduction and S-carboxymethylation, the fibrinogen polypeptide chains in each chromatographic peak were separated by ion exchange chromatography on DEAE-Sephacel and identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Aα, Bβ and smallest γ-chain (γ₅₀) eluted at progressively higher ionic strengths, but the elution positions of Aα, Bβ and γ₅₀ chains were identifcal for fibrinogen from each of the three different chromatographic fractions. The unique γ chain of fibrinogen in the second chromatographic peak (γ₅₅) eluted at an ionic strength higher than that of the γ₅₀ chain, while the largest γ-chain (γ_57.5), which was contained only in the third chromatographic peak of fibrinogen, eluted at the highest ionic strength. The higher ionic strengths needed to elute fibrinogen in the second and third peaks was paralleled by the higher ionic strengths needed to elute the γ-chains unique to them, suggesting that the γ-chain composition of the three fibrinogen fractions accounted for their differential binding to the ion exchange resin. Following desialation with neuraminidase, the differences in electrophoretic mobilities between the three γ-chain forms was maintained, indicating that differential migration on SDS-polyacrylamide gel electrophoresis was not due to variation in sialic acid content.     

Hemoglobin alpha-chain variation in macaques:primary structures of the alpha 1 and alpha II chains from the adult hemoglobins of Malaysian Macaca nemestrina.

The complete primary structures of each of the two α chains commonly found in the adult hemoglobins of Malaysian Macaca nemestrina (pig-tailed macaques) were obtained from the intact chains and five fragments produced by two nonenzymatic and three enzymatic cleavage reactions. The two chains differ at a single site, the α^I chains having a glutaminyl residue and the α^II chains having a histidinyl residue in position 78. Both chains differ from their human counterpart at five positions, the extent of divergence being similar to that observed for most of the α chains from other species of Macaca that have been analyzed to date. Elucidation of the structural difference between the α^I and α^II chains demonstrates that the high degree of heterogeneity observed among the hemoglobin phenotypes of M. nemestrina is a consequence of underlying genetic variability and not a result of postsynthetic modification of genetically identical proteins. Comparisons with the hemoglobin phenotypes found in other species of Macaca support the contention that chromosomes bearing linked α^I and α^II genes, as well as those bearing single α^I and α^II alleles, combine in zygotes to produce the phenotypic variation observed in members of Malaysian populations of M. nemestrina.     

Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives.

The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.     

Assay of proteins in the presence of interfering materials.

Glycinamidation of aminoethylated α, β, and γ chains of adult and fetal human hemoglobins results in the attachment of a glycinamide residue to each free carboxyl group. The peptides which result from tryptic hydrolysis of such modified chains can be separated by column chromatography or filter-paper finger-printing. Glycinamidation improves the yield of several tryptic peptides and is useful in determining the number and position of aspartyl and glutamyl residues in hemoglobins.     

High pK value of the N-terminal amino group of the gamma-chain causes low CO2 binding of human fetal hemoglobin.

The N-terminal amino acid residue of the γ-chains of human fetal hemoglobin (Hb F_II) is glycine rather than valine like in many other hemoglobins including the human adult pigment (Hb A). In the course of an evaluation of functional implications associated with this replacement we have studied the CO₂ binding properties of Hb F_II in comparison with Hb F_Ic where the N-termini of the γ-chains are blocked. By comparing Hb F_II and Hb F_Ic it is possible to specifically estimate carbamate formation at the N-termini of the γ-chains in Hb F_II. These data were used to calculate the carbamate equilibrium and ionization constant of these groups. At 37 °C, ?log₁₀ of the ionization constant (pK_z) was found to be 8.1 and is thus significantly higher than pK_z of the N-terminal valines of the β-chains of Hb A which has been reported to be 6.6 at 37 °C. The high pK_z value of the γ-chain α-amino group explains the much lower carbamate formation in Hb F_II compared to Hb A.     

Embryonic hemoglobins of different animal species

Summary The existence of embryonic hemoglobins is demonstrated in sheep-, calf and pig embryos. The occurrence and disappearance of these hemoglobins is quantitatively determined by cellulose acetate gel electrophoresis; hemoglobins as well as the globin chains, dissociated in 8 molar urea were quantitated. Sedimentation and diffusion experiments in the analytical ultracentrifuge revealed a S₂₀ of 4.3 and a D₂₀ of 6.6 for the examined hemoglobins. Therefore it is concluded that all hemoglobins occurring at different stages of embryonic and fetal development consist of 4 polypeptide chains with a total molecular weight of 66,000. The subsequent formation of the different polypeptide chains during ontogenesis is shown: At first only -chains are formed as demonstrated by the existence of Hb Gower I, consisting of four identical -chains. Subsequently the -chain appears, which leads to Hb Gower 2 (₂₂). The third polypeptide chain formed during the ontogenesis the -chain results finally in the appearance of HbF.In addition the existence of a HbF pig is demonstrated by the fingerprint technique.     

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.     

Complete amino acid sequences of the major early embryonic alpha-like globins of the chicken

Vertebrate embryos contain hemoglobins composed of globin polypeptides structurally distinct from those of adults. Together with fetal and adult globin chains, these early embryonic globins are encoded by two developmentally regulated multigene families. To facilitate analysis of the structure and evolution of early embryonic alpha-globin genes, we have determined the complete amino acid sequences of the pi and pi' alpha-like globins of the chick embryo. While differing from each other by an alanine/glutamic acid interchange at position 124, this pair of sequences differs from the major and minor adult alpha-globins by 43%. The early embryonic and adult alpha-like sequences appear to have diverged following an ancient gene duplication. We discuss specific amino acid substitutions in functional positions as possible mediators of the reduced Bohr effect and elevated oxygen affinity, which are characteristic of early embryonic hemoglobins.