Characterization of the hemoglobins and globins of Synbranchus marmoratus Bloch, 1795 (Pisces, Synbranchidae)

Total hemolysates of Synbranchus marmoratus Bloch, 1795, captured in Vitoriana, district of Botucatu, State of S?o Paulo, Brazil, were submitted to agar-starch gel electrophoresis on glass slides using 42 mM-Tris 1.7 mM EDTA-6.1 mM borate buffer, pH 8.8, for the gel and 10 mM borate-1.7 mM NaOH buffer, pH 8.6, for the cuvette. Three distinct hemoglobin bands were detected, with Hb I being of the cathodic type. Cellulose acetate electrophoresis in 800 mM Tris-2.1 mM EDTA buffer, pH 8.9, containing 6 M urea and 2.25 mM beta-mercaptoethanol indicated the presence of four globin chains denoted alpha 1, alpha 2, beta and gamma. It is suggested that the probable tetrameric constitution of the hemoglobin of Synbranchus marmoratus Bloch, 1795 is Hb I (alpha (2)2 gamma 2), Hb II (alpha (1)2 gamma 2) and Hb III (alpha (1)2 beta 2).     

The primary structure of the hemoglobins of the adult jaguar (Panthera onco, Carnivora)

The primary structure of the hemoglobins from Jaguar (Panthera onco) are presented. Electrophoretic separations without and with a dissociating agent revealed the presence of two hemoglobin components, alpha 2 beta I2 and alpha 2 beta II2. The separation of the hemoglobin components was achieved by ion-exchange chromatography. The globin chains were separated by ion-exchange chromatography and also by reversed phase HPLC. The amino-acid sequences of the native chains and peptides were determined by liquid-phase and gas-phase sequencing. N-Acetylserine was detected by FAB-mass spectroscopy as N-terminal group of the beta I chain. The sequences are compared with that of human hemoglobin (Hb A).     

Differential expression of Iα- and IIα-globin genes in Norwegian dairy goats

Summary. Fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis were used to determine the ratio between ^Iα-and ^IIα-globin chains in Norwegian dairy goats. Three different phenotypes, designated normal (N) with ^Iα- to ^IIα-globin ratio 3:1, reversed (R) with ratio 1:2 and double-reversed (RR) with no ^Iα-globin, were described. Family studies indicated that the R animals were heterozygous, and the RR animal homozygous, for a haplotype without a functional ^Iα-globin product. Southern blot analysis of goat DNAs digested with six different restriction enzymes showed that the different ratios of α chain expression could not be due to a deletion of the ^Iα-gene and/or duplication or triplication of the ^IIα-globin genes. The homozygous reversed animal with no detectable ^Iα-globin had a mild anaemia.     

The primary structures of the major and minor hemoglobin-components of adult Andean goose (Chloephaga melanoptera, Anatidae): the mutation Leu----Ser in position 55 of the beta-chains

The primary structures of the hemoglobin components Hb A and Hb D of the adult Andean Goose (Chloephaga melanoptera) are presented. The globin chains were separated on CM-Cellulose in 8M urea buffer. The amino-acid sequences were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid- and gas-phase sequenators. The sequences are aligned with those of Greylag Goose (Anser anser) as a biological reference and other sequences of birds. A detailed evaluation of all residues of Andean Goose hemoglobins on the basis of the 12000 known avian globin sequences leads to a molecular pattern for high-altitude respiration of geese. The replacement of functional and structural importance is the unique occurrence of the residue beta 55 Leu----Ser (all other exchanges are functionally neutral), interrupting the same alpha 1 beta 1-interface contact (alpha 119-beta 55) that accounts for high-altitude respiration of the Barheaded Goose (Anser indicus); there the mutation is found on alpha A 119. Loosening the constraints of this interface must be interpreted as a destabilization of the low-affinity T-structure in favour of the high-affinity R-structure. The structural and functional significance of this interface for the molecular biology of high-altitude respiration of the Andean Goose and Barheaded Goose is discussed. Since Hb A consists of alpha A2 beta 2 and Hb D consists of alpha D2 beta 2 the mutation occurring in blood of the Andean Goose affects both hemoglobins whereas in the case of the Barheaded Goose only Hb A is affected. These results show that Hb D can be considered a biological reserve to enlarge situatively the normal hemoglobin function. A general molecular pattern for permanent (selective advantage of high intrinsic oxygen affinity) and transitory (selective advantage of graded oxygen affinities) adaptation to hypoxia is discussed. A survey on the sequence homology of the globin chains of geese (Anserinae) and ducks (Anatinae) is given.     

Haemoglobin phenotypes of the wild European mouflon sheep living on the island of Sardinia

Haemoglobin (Hb) phenotypes have been studied in 100 wild European mouflons living on the island of Sardinia by means of isoelectric focusing (pH 6.7-7.7 range) of the native tetramers, acid-urea-Triton gel-electrophoresis, and reversed-phase HPLC of globin chains. The result indicates the presence of two beta-globin alleles one of which, corresponding to the beta B, being the most common (f = 0.94). None were carriers of the earlier described Hb A. The new Hb was provisionally named Hb M. Severely anaemic mouflons were able to synthesize Hb C at expense of the Hb B alone, thus suggesting structural and physiological homologies between mouflon beta B and sheep beta A globin genes, and between the newly observed beta M allele and the beta B of the domestic Sardinian sheep.     

The stability of the heme-globin linkage in some normal, mutant, and chemically modified hemoglobins.

The rates and equilibria of heme exchange between methemoglobin and serum albumin were measured using a simple new spectrophotometric method. It is based on the large difference between the spectrum of methemoglobin and that of methemealbumin at pH 8-9. The rate of heme exchange was found to be independent of the albumin concentration and inversely proportional to the hemoglobin (Hb) concentration. Taken together with the finding that the rate was 10 times greater for Hb Rothschild, which is completely dissociated into alpha beta dimers and 10 times smaller for two cross-linked hemoglobins, the subunits of which cannot dissociate, this showed that the rate of dissociation of heme from alpha beta dimers is very much greater than from tetramers. Conditions were found for the attainment of an equilibrium distribution of hemes between beta globin and albumin. The equilibrium distribution ratio, R = methemealbumin/albumin/methemoglobin/apohemoglobin, for hemoglobin A was 3.4 with human and 0.005 with bovine serum albumin. Both the rates of exchange and the R values of HbS and HbF were the same as that for HbA. The equilibrium distribution ratio for Hb Rothschild was 7 times greater than that for HbA whereas that of one but not the other of the cross-linked hemoglobins was 10 times smaller. The structural bases for these differences are analyzed.     

应用实时定量RT-PCR技术检测b地中海贫血 珠蛋白基因的表达

为了定量检测 b 地中海贫血(b 地贫)的 a、b 和γ珠蛋白基因表达水平, 提取正常成人对照组、正常胎儿对照组和b 地贫患者组组成的样本 DNA,采用反向点杂交法（RDB）分析b 地贫各种突变类型;提取样本RNA用于进行针对a、b 和γ珠蛋白基因的荧光实时定量RT-PCR（FQ RT-PCR）。根据FQ RT-PCR原理,设计合成分别对应于a、b 和γ珠蛋白基因的3对引物和3条荧光探针,FQ RT-PCR在ABI 7700系统进行。用SPSS 10.0对实验数据进行统计学分析,分别计算正常对照组 (bA/bA,aa/aa),脐带血组(bA/bA,aa/aa),轻型b 地贫组(bT/bA,aa/aa),重型b地贫组(bT/bT,aa/aa)的a、b 和γmRNA比值,其中a/b分别为4.62±1.20、7.81±2.89、13.51±5.12、188.24±374.04;a/(b +γ)分别为4.43±1.17、0.56±0.49、9.62±4.37、2.14±1.58;γ/(b+γ) 分别为0.04±0.03、0.92±0.06、0.28±0.18、0.95±0.04。由于组与组之间均值变异范围较大,将其进行对数转换后再进行方差分析。结果表明： a/b与a/(b+γ)在所有组与组之间均有显著性差异。γ/(b+γ)除了在脐带血组和重型b地贫组之间无显著性差异外,在其他组与组之间均有显著性差异。实验说明,人类b珠蛋白基因的表达水平从正常对照组到重型b地贫组急剧下降且以重型b地贫组为最低;相反γ珠蛋白基因表达却明显升高,以重型b地贫组为最高。与正常成人对照组相比,胎儿期b mRNA水平较低但γmRNA 水平较高。因此,正常个体不同时期和不同类型b 地贫之间a、b与γ珠蛋白基因表达不同而且互相影响。 Abstract：whole blood samples were collected from 100 normal healthy adults, from umbilical cord of 33 newborn infants, 111 individuals with b-thalassemia minor (bT/bA,aa/aa) and 39 with b-thalassemia major (bT/bT,aa/aa). Prior to quantitative analysis of globin gene expression, DNA was extracted from all blood samples and used for b-thalassemia genotype analysis. Different types of b globin gene mutations were analyzed using reverse dot blotting (RDB) method. Total RNA were extracted and subjected to real-time RT-PCR for quantitative measurement of a, b andγglobin mRNA using three sets of primers and fluorescent-labeled probes, designed according to the sequences of a, b andγhuman globin gene. Real-time RT-PCR was performed in ABI 7700 system. Following the real-time RT-PCR, the mean values of a, b andγglobin mRNA were calculated and the ratios of a/b, a/(b + γ) andγ/(b + γ) were determined to characterize the relative expression levels of different globin genes among normal adult, infant, b-thalassemia minor and b-thalassemia major patients. The resultant data were analyzed using SPSS 10.0 software to determine statistical significance of human globin gene expression among normal controls and b-thalassemia patients. Due to vast variations of the mean globin gene mRNA levels among different groups, log conversion of a/b + 1, a/(b + γ) + 1 andγ/(b + γ) +1 was used for statistical analyses and intergroup comparison. The a/b globin gene mRNA ratios were determined to be 4.62±1.20, 7.81±2.89, 13.51±5.12, and 188.24±374.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b-thalassemia major(bT/bT,aa/aa) respectively. The a/(b+γ) ratios were 4.43±1.17, 0.56±0.49, 9.62±4.37, and 2.14±1.58 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Theγ/(b+γ) ratios were 0.04±0.03, 0.92±0.06, 0.28±0.18, and 0.95±0.04 for normal healthy adult (bA/bA,aa/aa), infant (bA/bA,aa/aa), b- thalassemia minor (bT/bA,aa/aa) and b- thalassemia major(bT/bT,aa/aa) respectively. Following statistical analyses, the a/b and a/(b+γ) globin gene mRNA ratios were significantly different among four different groups (normal adult, normal infant, b- thalassemia minor and b- thalassemia major). The γ/(b + γ) globin gene mRNA ratio was significantly different among all groups except for between infant and b- thalassemia major patients. Human b globin gene mRNA levels decrease progressively and dramatically from normal adults to b-thalassemia patients with b-thalassemia major having the lowest levels. On the other hand, the γglobin gene mRNA levels increase progressively from normal adult to b-thalassemia patients with b-thalassemia major having the highest levels. Infants have relatively lower levels of b but higher levels of γglobin gene mRNA as compared to those in normal adults. Thus, the relative expression levels of a, b or γglobin genes varied but inter-related among different ages of normal individuals and different b-thalassemia genotypes.     

Relative proteome quantification of alpha,beta, gamma and delta globin chains in early eluting peaks of Bio-Rad variant II® CE-HPLC of hemoglobin from healthy and beta-thalassemia subjects in Malaysia

This is the first report of QQQ-mass spectrometric identification and quantification of the Hb subunits, alpha, beta, delta and gamma globin peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the Bio-Rad cation-exchange chromatography of haemoglobin. The objectives were to assess the relationship of the quantity of the free alpha, beta, delta and gamma globin chains with the phenotypic diversity of beta-thalassaemias (β-thal). The results demonstrate that the pools of free globin chains in red blood cells were correlating with the severity of the disease in patients with different phenotypes of β-thal. The mechanism and the regulation of synthesis of free globin chains pool in a normal individual and in patients with different β-thal phenotypes could arise from several mechanisms which will require further investigation. The role of the free globin pool in patients with β-thal for development of novel therapeutic approaches based on these potential targets requires further investigation. Pertinent biomarkers improves the diagnosis of the β-thal, especially in low-income countries where they are most common and allows more effective therapeutic intervention leading to more successful therapeutic outcome.     

Sickle cell-beta +-thalassaemia: a haematological and clinical study in Liberia

Clinical and haematological features of 20 patients of several Liberian ethnic groups with sickle cell-beta +-thalassaemia are reported. Haemoglobin analysis showed increased Hb A2 values, high Hb A levels (median 25%), variable amounts of Hb F and a slight imbalance of non alpha/alpha globin chain synthesis ratios. The clinical and other haematological findings varied but the disease seems to run a relatively mild course in the majority of the patients.     

High-altitude respiration of birds. Structural adaptations in the major and minor hemoglobin components of adult Rüppell's Griffon (Gyps rueppellii, Aegypiinae): a new molecular pattern for hypoxic tolerance

The primary structures of the hemoglobins Hb A, Hb A', Hb D and Hb D' of Rüppell's Griffon (Gyps rueppellii), which can fly as high as 11,300 m, are presented. The globin chains were separated on CM-Cellulose in 8M urea buffers, the four hemoglobin components by FPLC in phosphate buffers. The amino-acid sequences of five globin chains were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid-phase and gas-phase sequenators. The sequences are compared with those of other Falconiformes. A new molecular pattern for survival at extreme altitudes is presented. For the first time four hemoglobins are found in blood of a bird; they show identical beta-chains and differ in the alpha A- and alpha D-chains by only one replacement. These four hemoglobins cause a gradient in oxygen affinities. The two main components Hb A and Hb A' differ at position alpha 34 Thr/Ile. In case of Ile as found in Hb A' an alpha 1 beta 1-interface is interrupted raising oxygen affinity compared to Hb A. In addition the hemoglobins of the A- and D-groups differ at position alpha 38 Pro or Gln/Thr (alpha 1 beta 2-interface). Expression of Gln in Hb D/D' raises the oxygen affinity of these components compared to Hb A/A' by destabilization of the deoxy-structure. The physiological advantage lies in the functional interplay of four hemoglobin components. Three levels of affinity are predicted: low affinity Hb A, Hb A' of intermediate affinity, and high affinity Hb D/D'. This cascade tallies exactly with oxygen affinities measured in the isolated components and predicts oxygen transport by the composite hemoglobins over an extended range of oxygen affinities. It is contended that the mechanisms of duplication of the alpha-genome (creating four hemoglobins) and of nucleotide replacements (creating different functional properties) are responsible for this remarkable hypoxic tolerance to 11,300 m. Based on this pattern the hypoxic tolerances of other vultures are predicted.     

Contribution of alpha140Tyr and beta37Trp to the near-UV CD spectra on quaternary structure transition of human hemoglobin A

The CD band of human adult hemoglobin (Hb A) at 280 approximately 290 nm shows a pronounced change from a small positive band to a definite negative band on the oxy (R) to deoxy (T) structural transition. This change has been suggested to be due to environmental alteration of Tyrs (alpha42, alpha140, and beta145) or beta37 Trp residues located at the alpha1beta2 subunit interface by deoxygenation. In order to evaluate contributions of alpha140Tyr and beta37Trp to this change of CD band, we compared the CD spectra of two mutant Hbs, Hb Rouen (alpha140Tyr-->His) and Hb Hirose (beta37Trp-->Ser) with those of Hb A. Both mutant Hbs gave a distinct, but smaller negative CD band at 287nm in the deoxy form than that of deoxyHb A. Contributions of alpha140Tyr and beta37Trp to the negative band at the 280 approximately 290 nm region were estimated from difference spectra to be 30% and 26%, respectively. These results indicate that the other aromatic amino acid residues, alpha42Tyr and beta145Tyr, at the alpha1beta2 interface, are also responsible for the change of the CD band upon the R-->T transition of Hb A.     

Chemical evidence for the separation of G gamma and A gamma chains by polyacrylamide gel electrophoresis in urea and triton X-100

Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: alpha, beta, and 2 gamma globin chains. We have verified that the latter two are the G gamma and A gamma globin chains which have respectively glycine or alanine at position 136. After incorporation of either [3H] alanine or [3H] glycine into newly synthesized globin each gamma chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incorporation was detected for the third turn (position 136) of the G gamma chain and alanine for the A gamma. Substantial metabolic conversion of [3H] glycine to serine and proline was also noted.     

Effect of polyamines on hemoglobin and globin chain synthesis

Exposure of reticulocytes to 25 mM spermidine and spermine stimulates the incorporation of 14C leucine into hemoglobin and globin polypeptide chains. These two polyamines stimulate beta chain synthesis to a greater extent than they stimulate alpha chain synthesis, thus resulting in a decreased synthetic alpha/beta globin ratio. Because of the low permeability of the red cell membrane to these polycations, less than 1.0% of extracellular spermidine and spermine accumulate within the erythrocyte, thus these effects occur at intracellular polyamine levels that are physiological. Putrescine progressively decreases hemoglobin synthesis at extracellular concentrations greater than 10 mM without affecting the synthetic alpha/beta ratio. These findings suggest a role for polyamines in the fine tuning of the alpha/beta globin ratio at the translational level.     

Characteristics in tyrosine coordinations of four hemoglobins M probed by resonance Raman spectroscopy

Resonance Raman spectra of four hemoglobins (Hbs) M with tyrosinate ligand, that is, Hb M Saskatoon (beta distal His----Tyr), Hb M Hyde Park (beta proximal His----Tyr), Hb M Boston (alpha distal His----Tyr), and Hb M Iwate (alpha proximal His----Tyr), were investigated in order to elucidate structural origins for distinctly facile reducibility of the abnormal subunit of Hb M Saskatoon in comparison with other Hbs M. All of the Hbs M exhibited the fingerprint bands for the Fe-tyrosinate proteins around 1600, 1500, and 1270 cm-1. However, Hb M Saskatoon had the lowest Fe-tyrosinate stretching frequency and was the only one to display the Raman spectral pattern of a six-coordinate heme for the abnormal beta subunit; the others displayed the patterns of a five-coordinate heme. The absorption intensity of Hb M Saskatoon at 600 nm indicated a transition with a midpoint pH at 5.2, whereas that of Hb M Boston was independent of pH from 7.2 to 4.8. The fingerprint bands for the tyrosinate coordination as well as the Fe-tyrosinate stretching band disappeared for Hb M Saskatoon at pH 5.0, and the resultant Raman spectrum resembled that of metHb A, while those bands were clearly observed for Hb M Boston at pH 5.0 and for two Hbs M at pH 10.0. These observations suggest that the unusual characteristics of the heme in the abnormal beta chain of Hb M Saskatoon result from the weak Fe-tyrosinate bond, which allows weak coordination of the proximal histidine, giving rise to the six-coordinate high-spin state at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sites of modification of hemospan, a poly(ethylene glycol)-modified human hemoglobin for use as an oxygen therapeutic

Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW approximately 5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of approximately 8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual alpha and beta globins was made using reverse-phase HPLC, showing a series of alpha globins conjugated with 0 to 3 PEGs and a series of beta globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on beta globins at beta8, beta17, beta59, beta66, beta93, beta95, and beta132 and three sites identified on alpha globins at alpha7, alpha16, and alpha40.     

Tangential flow filtration of hemoglobin

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS‐PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P₅₀) and cooperativity coefficient (n) were regressed from the measured oxygen‐RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC‐MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α‐ and β‐ globin chains. Taken together, our results demonstrate that HPLC‐grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb‐based oxygen carriers. © 2008 American Institute of Chemical Engineers, 2009     

Structural analysis of the minor human hemoglobin components: Hb AIa1, Hb AIa2 and Hb AIb

Human hemolysate contains several minor hemoglobin components, including Hb AIa1, Hb AIa2, Hb AIb and Hb AIc which are post-translational modifications of the major component, Hb A0. Hb AIc is known to contain glucose attached to the N terminus of the beta chains by a ketoamine linkage. We separated the alpha and beta globin chains from purified Hb AIa1, Hb AIa2 and Hb AIb by ion-exchange chromatography. The beta chains were reducible by sodium borohydride and gave a positive thiobarbituric acid test. These results indicated that they are modified by ketoamine-linked carbohydrate. In addition, phosphate analysis revealed 1.5 phosphate residue associated with each beta AIa1 chain and 1 phosphate residue with each beta AIa2 chain. Hb AIa1, Hb AIa2 and Hb AIb were all found to be contaminated by non-globin proteins. Protein-sequencing approaches demonstrated that the N termini of beta AIa1, beta AIa2 and beta AIb were blocked. In support of this conclusion, analysis of tryptic digests of beta AIa2 and B AIb revealed modified N-terminal peptides. We conclude that, like Hb AIc, components Hb AIa1, Hb AIa2 and Hb AIb also contain a sugar moiety linked to the N terminus of the beta chain.