Analysis of the globins from fast human haemoglobins by isoelectrofocusing on polyacrylamide gel rods

The globins from all fast haemoglobin (Hb) components obtainable by Bio-Rex 70 cation-exchange chromatography were examined by isoelectrofocusing on polyacrylamide gel rods with 8.0 mol/l urea. From this analysis HbA1a1 and HbA1a2 seem to be very heterogeneous components. HbA1b is separable into two components, one of which is varied in both the beta chains. Between HbA1b2 and the well-known HbA1c components two chromatographic peaks are separated, one with a noticeable percentage of glucosylated beta chain and one that probably contains HbF. HbA1c has both beta chains glucosylated, while HbA1x seems to be a beta monoglucosylated Hb form. Finally, the early part of the HbAo peak has a large amount of glucosylation on both alpha and beta chains.     

Reversed-phase high-performance liquid chromatography of human haemoglobin chains

A reversed-phase high-performance liquid chromatographic method for the separation of human haemoglobin chains has been devised. Using a LiChrospher 100 CH-8/2 column and a ternary eluent (acetonitrile-methanol-0.155 M NaCl, pH 2.7) improved resolution was achieved between (delta beta) Lepore, beta A, beta S, alpha, G gamma and A gamma chains within a 60-min linear gradient. The A gamma T chain can also be separated by increasing the gradient time and decreasing the flow-rate. Silanophilic interactions play an important role in the retention mechanism, and NaCl addition was necessary in order to suppress adsorption on free silanols. Increasing the methanol concentration to 10% caused a slight increase in chain retention, probably owing to solvation of the stationary phase. The recovery was 82% and the reproducibility of retention times was as good as +/- 1.5%. Quantitation of chains is likely to be possible by peak area measurement. Owing to its sensitivity, the proposed method may be useful in the diagnosis of haemoglobinopathies and in the study of haemoglobin variants.     

Quantitation of hemoglobins Bart's, H, Portland-I, Portland-II and constant spring by anion-exchange high-performance liquid chromatography

We introduce a new high-performance liquid chromatographic procedure that uses a specific anion-exchange column for the separation of hemoglobin (Hb) Bart's (gamma 4), Hb H (beta 4), Hb Portland-I (zeta 2 gamma 2), Hb Portland-II (zeta 2 beta 2), and the abnormal Hb Constant Spring (alpha 2 extended beta 2) in cord blood and adult red cell lysates. The method provides quantitative data for Hb Bart's in cord blood that correlate well with the alpha-globin gene status of the babies and can be used for an initial identification of alpha-thalassemic conditions. Quantitation of Hb Bart's from cord blood samples that are collected on filter paper and submitted as dried blood spots is unreliable. The separation of Hb H and Hb Bart's allows an evaluation of the synthesis of these two hemoglobin components in patients with Hb H disease.     

Negative ion fragmentations of deprotonated peptides: backbone cleavages directed through both Asp and Glu

The collision-induced spectra of [M - H](-) ions of a variety of natural and synthetic amphibian peptides containing Asp and/or Glu exhibit characteristic gamma backbone cleavage ions that identify the positions of these residues in the peptide. A theoretical study suggests that the Glu cleavage involves an S(N)i reaction of the carboxylate anion from the Glu alpha side chain to form a deprotonated cyclic lactone. The presence of either Asp or Glu or other residues that effect pronounced side-chain cleavages (e.g. Ser or Thr) results in the normal alpha and beta backbone cleavages being reduced in comparison to those cleavages which originate from side chains.     

Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focusing method

To analyze both hemoglobin (Hb) and globin chain variants, we modified a commonly used method, capillary isoelectric focusing (CIEF), with detection at 280 nm. The samples were hemolysates prepared from red blood cells, and globin chains obtained from the hemolysates by treatment with cold acidified acetone. When the migration time for the internal reference, carbonic anhydrase I (isoelectric point, pI 6.60), was taken as 1.0, the migration ratio for Hb A0 in normal human blood was 0.877 +/- 0.004 (mean +/- SD, n = 9), and those of the alpha- and beta-globin chains were 0.673 +/- 0.004 and 0.847 +/- 0.005 (mean +/- SD, n = 4), respectively. The ratio of peak heights between the beta- and alpha-globin chains (beta/alpha) in the normal Hbs obtained from four subjects was almost constant at 2.5 +/- 0.1 (mean +/- SD). This ratio indicates which of the globin chains includes a mutation (if one exists). When an Hb variant, Hb Hoshida (in which Gln is substituted for Glu at residue 43 in the beta-globin chain), was analyzed by this method, two main peaks were observed (migration ratios 0.836 and 0.877, corresponding to an abnormal and the normal Hb, respectively). An additional peak with an abnormal migration ratio of 0.788 was also detected in the globin chain profiles. The ratio of peak heights between normal beta- and alpha-globin chains was 1.57, indicating that a mutation exists in the beta-globin chain. We thus established a convenient system using CIEF that provides a rapid and reproducible method for the random analysis of both Hb and globin chain variants.     

Molecular modeling and mutagenesis reveals a tetradentate binding site for Zn2+ in GABA(A) alphabeta receptors and provides a structural basis for the modulating effect of the gamma subunit

Gamma-aminobutyric acid type A receptors (GABA(A)-R) containing alpha1beta2gamma2 subunits are weakly inhibited by Zn2+, whereas receptors containing only the alpha1beta2 subunits are strongly inhibited. We built homology models of the ion pores of alpha1beta2 and alpha1beta2gamma2 GABA(A)-R using coordinates of the nicotinic acetylcholine receptor as a template. Threading the GABA(A)-R beta2 sequence onto this template placed the 17' histidine and the 20' glutamate residues at adjacent locations in the mouth of the pore, such that a nearly ideal tetradentate site for Zn2+ was formed from two histidine and two glutamate residues between adjacent beta subunits in the alpha1beta2 GABA(A)-R. Following optimization with CHARMM, the distance between the alpha-carbons of the adjacent histidine residues was approximately 9.2 A, close to the ideal distance for a Zn2+ binding site. Loss of inhibition by Zn2+ in alpha1beta2gamma2 GABA(A)-R can be explained by the geometry of these residues in the arrangement alpha1beta2gamma2alpha1beta2, in which the nearest C-alpha-C-alpha distance between the histidine residues is 15.5 A, too far apart for an energetically optimal Zn2+ binding site. We then mutated the gamma subunit at the 17' and/or 20' positions. Zn2+ inhibition was not restored in alpha1beta2gamma2 (I282H) receptors. A novel finding is that the modeling shows the native 20' lysine in gamma2 can compete with Zn2+ for binding to the inserted 17' histidine. Sensitivity to Zn2+ was restored in the double mutant receptor, alpha1beta2gamma2 (I282H; K285E), in which the competition with lysine was removed and a more favorable Zn2+ binding site was formed.     

Detection of the embryonic zeta chain in blood from newborn babies by reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) using the large-pore Vydac C4 column has been used to detect and quantitate the embryonic zeta chain in blood samples of normal babies and of newborns with varying degrees of alpha chain deficiencies. The zeta chain eluted at the end of the chromatogram at about 130 min using a modified and extended gradient. Its identity was confirmed by structural analysis of zeta chain isolated from a blood sample of a fetus without active alpha globin genes, i.e. with hydrops fetalis (--/--). The quantity of zeta in normal babies is less than 0.7% [% of (alpha + zeta)] and is dependent upon the maturity of the baby as it was only present in babies with low levels of beta chain or hemoglobin (Hb) A. The presence of a zeta globin gene deletion [A. E. Felice et al., Hum. Genet., 73 (1986) 221; and P. Winichagoon et al., Nucleic Acids Res., 10 (1982) 5853] did not affect the level of zeta in the newborn. All babies with an alpha-thalassemia-2 heterozygosity, i.e. with three active alpha globin genes or -alpha/alpha alpha, had zeta in a range of 0.1-0.9%; again the level showed a negative correlation with that of the beta chain. Newborns with an alpha-thalassemia-2 homozygosity or -alpha/-alpha had a varying level of zeta of 0.3-2.3%, which did not correlate with the level of beta, suggesting that zeta chain production persists after birth in this condition. Macrochromatographic analyses in combination with RP-HPLC indicated that the zeta chain is present as zeta 2 gamma 2 or Hb Portland-I, as expected.     

Quantities of adult, fetal and embryonic globin chains in the blood of eighteen- to twenty-week-old human fetuses

The prenatal diagnostic program, established at Hacettepe University in Ankara for the purpose of detecting beta-thalassemia (beta-thal), sickle cell anemia (SS), and Hb S-beta-thal, offered the opportunity of evaluating the relative quantities of adult (beta A, beta S), fetal (G gamma, A gamma, A gamma T), and embryonic (epsilon, zeta) chains in 26 fetuses, aged 18-20 weeks. Methodology involved micro high-performance liquid chromatographic (HPLC) procedures and immunology using an mAb, specific for the embryonic epsilon chain. A good correlation was observed between the beta/gamma in vitro chain synthesis ratio and the level of beta A and/or beta S chains determined by reversed-phase HPLC; the combination of these two sets of data strengthens the prenatal diagnostic approach of detecting beta-thal major but not beta-thal trait. The levels of the different gamma chains were about as observed in newborn babies; the frequency of the A gamma T variant in the 26 fetuses was the same as observed for a larger group of Turkish newborn babies. The level of the embryonic zeta chain was higher than seen in full-term babies and varied between 0 and 1.3%; 5 of the 26 fetuses showed the complete absence of zeta. The embryonic epsilon chain was not detectable, not even in babies with beta-thal major. These data indicate that the synthesis of epsilon is completely turned off in fetuses at the age of 18-20 weeks, while that of zeta continues, albeit at a low level.     

Quantitation of methanol formed in cell culture cytotoxicity assays and as a metabolite in microsome suspensions

Two methods have been used to isolate Hb F from red cells with low levels of Hb F (less than 2% FAD), namely an alkali denaturation procedure, as described by Tsuchiya et al. [Dokkyo J. Med. Sci., 10 (1983) 13], and anion-exchange chromatography. Analyses of these Hb F enriched hemoglobin solutions by high-performance liquid chromatography allowed quantitation of the different gamma chains in the Hb F. Although the data showed considerable variation, particularly for samples with low levels of Hb F, the final results obtained with the two approaches were comparable, suggesting that the much simpler and more economical alkali denaturation procedure can be used for this purpose.     

Negative ion electrospray mass spectra of caerulein peptides: an aid to structural determination

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH(2)O), Thr (-CH(3)CHO) and Asp (-H(2)O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert.     

Simplified hemoglobin chain detection by capillary electrophoresis

Hemoglobin (Hb) chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb chains also after sample extraction. We describe a CE method for analysis of Hb chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the chains were denatured and separated from the heme during electrophoresis. The common variants of the beta-chains, such as beta(S), beta(C), and beta(E), are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the hemoglobinopathies.     

聚磷链和聚氮链的自洽场晶体轨道研究

利用自治场全略微分重叠晶体轨道方法(SCFCNDO/2-CO)对聚磷链和聚氮链的各种可能构型的稳定性、能带结构和电子特性进行了研究.结果表明,本征聚磷链为半导体,而本征聚氮链为绝缘体.     

Chromatographic analysis of tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle.

Tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle all exist as dimeric, ca. 100% alpha-helical coiled-coil species in benign media. Two major tropomyosin isoforms from each muscle source have been identified and can be conveniently designated alpha (fast) and beta (slow) based on electrophoretic mobility under denaturing conditions. The ratio of alpha to beta chains is ca. 3-4:1 for rabbit skeletal and ca. 1:1 for chicken gizzard and earthworm tropomyosins. Each chain from the former two muscle sources has been sequenced, thus providing a molecular basis for interpreting the in vivo population of homo- and hetero-dimers. The characteristics of each purified tropomyosin in weak-anion exchange, strong-cation exchange and reversed-phase high-performance liquid chromatography are described. Binding to and/or elution from the reversed-phase matrix results in dissociation into highly helical monomeric chains. This mode of chromatography separates the alpha and beta chains of earthworm and chicken gizzard tropomyosins, but not those of the rabbit protein. Both anion- and cation-exchange chromatography use mild (benign) elution conditions under which the native, in vivo dimer population should be preserved. Only the rabbit protein exhibited peak separation on the anion-exchange resin, with peak assignment corresponding to the known molecular organization of homo- and hetero-dimers. In strong cation-exchange analysis, all three tropomyosins exhibit a chromatographic transition near pH 6.5, possibly the result of histidine(s) titration. Collectively, the chromatographic data confirm the present understanding of the in vivo mixture of dimers for tropomyosin from rabbit skeletal and chicken gizzard. It is concluded that native earthworm tropomyosin exists predominantly as an alpha beta hetero-dimer.     

Comparison of the positive and negative ion electrospray mass spectra of some small peptides containing pyroglutamate

The positive ion electrospray mass spectra of [M+H](+) and the negative ion electrospray mass spectra of [M-H](-) ions of selected pyroglutamate containing peptides both provide sequencing data. The negative ion spectra show the normal alpha and beta backbone cleavages in addition to delta and gamma backbone cleavages initiated by the side chains of Glu and Phe residues. For example, the [M-H](-) ion of pGlu Pro Gln Val Phe Val-NH(2) shows delta and gamma peaks at m/z 224 (delta, Gln3), 244 (gamma, Phe4), 451 (delta, Phe4), 471 (gamma, Gln3). Some of the negative ion spectra show unusual grandaughter peaks that originate by alpha and beta, or delta and gamma backbone cleavages of a beta(1) cleavage ion.     

The M gamma chain of human fetal hemoglobin; its identification and occurrence

High-performance liquid chromatographic procedures have been used in the detection and identification of a new gamma chain of human fetal hemoglobin (Hb). This M gamma chain is characterized by a Leu----Met replacement at position gamma 141; no other structural variations have been observed. The M gamma chain has been detected in red cell lysates of subjects with a heterozygosity for one of many types of so-called hereditary persistence of fetal hemoglobin conditions, which are characterized by an increased level of Hb F in adult life, in sickle cell anemia, and in a few cord blood samples. At present it is not possible to definitely identify the genetic cause of this newly discovered heterogeneity; an infidelity in translation or the existence of an unrecognized gamma globin gene should be considered.     

Importance of chirality and reduced flexibility of protein side chains: a study with square and tetrahedral lattice models

Side chains of amino acid residues are the determining factor that distinguishes proteins from other unstable chain polymers. In simple models they are often represented implicitly (e.g., by spin states) or simplified as one atom. Here we study side chain effects using two-dimensional square lattice and three-dimensional tetrahedral lattice models, with explicitly constructed side chains formed by two atoms of different chirality and flexibility. We distinguish effects due to chirality and effects due to side chain flexibilities, since residues in proteins are L residues, and their side chains adopt different rotameric states. For short chains, we enumerate exhaustively all possible conformations. For long chains, we sample effectively rare events such as compact conformations and obtain complete pictures of ensemble properties of conformations of these models at all compactness region. This is made possible by using sequential Monte Carlo techniques based on chain growth method. Our results show that both chirality and reduced side chain flexibility lower the folding entropy significantly for globally compact conformations, suggesting that they are important properties of residues to ensure fast folding and stable native structure. This corresponds well with our finding that natural amino acid residues have reduced effective flexibility, as evidenced by statistical analysis of rotamer libraries and side chain rotatable bonds. We further develop a method calculating the exact side chain entropy for a given backbone structure. We show that simple rotamer counting underestimates side chain entropy significantly for both extended and near maximally compact conformations. We find that side chain entropy does not always correlate well with main chain packing. With explicit side chains, extended backbones do not have the largest side chain entropy. Among compact backbones with maximum side chain entropy, helical structures emerge as the dominating configurations. Our results suggest that side chain entropy may be an important factor contributing to the formation of alpha helices for compact conformations.     

Molecular dynamics simulation of the glass transition temperature of fullerene filled cis-1,4-polybutadiene nanocomposites

t In this work,the effect of the fullerene (C60) weight fraction and PB-C60 interaction on the glass transition temperature (Tg) of polymer chains has been systemically investigated by adopting the united atom model of cis-1,4-poly(butadiene) (cis-PB).Various chain dynamics properties,such as atom translational mobility,bond/segment reorientation dynamics,torsional dynamics,conformational transition rate and dynamic heterogeneity of the cis-PB chains,are analyzed in detail.It is found that Tg could be affected by the C60 weight fraction due to its inhibition effect on the mobility of the cis-PB chains.However,Tg is different,which depends on different dynamics scales.Among the chain dynamics properties,Tg is the lowest from atom translational mobility,while it is the highest from the dynamic heterogeneity.In addition,Tg can be more clearly distinguished from the dynamic heterogeneity;however,the conformational transition rate seems to be not very sensitive to the C60 weight fraction compared with others.For pure cis-PB chains,Tg and the activation energy in this work can be compared with those of other polymers.In addition,the temperature dependence of the dynamic properties has different Arrhenius behaviors above and below Tg.The activation energy below Tg is lower than that above Tg.This work can help to understand the effect of the C60 on the dynamic properties and glass transition temperature of the cis-PB chains from different scales.     

Characterization of the elusive disulfide bridge forming human Hb variant: Hb Ta-Li β83 (EF7)Gly → Cys by electrospray mass spectrometry

An electrospray mass spectrometric approach to the identification of a human hemoglobin (Hb) variant involving a Cys residue incorporation is presented. In Hb Ta-Li (beta83Gly --> Cys), Cys83 forms inter-molecular disulfide bridges. Routine analysis of the denatured Hb showed the presence of a minor beta chain variant whose mass apparently was 1 Da less than the expected mass difference of 46 Da for a Gly --> Cys substitution. Reduction of the globin chains with dithiothreitol gave an intense monomer with the expected mass difference for the Gly --> Cys substitution. After reprocessing the original raw data from the denatured Hb and taking into account the possibility of dimer formation, a component was revealed whose mass was consistent with a disulfide linked dimer of Ta-Li beta globins. The mutation was localized to peptide betaT10 by analysis of a tryptic digest. Tandem mass spectrometry and DNA sequencing confirmed the Gly --> Cys substitution occurred at residue 83 of the beta chain. Problems encountered in identifying the components in mixtures of monomers and dimers are discussed.     

Backbone cleavages of [M - H](-) anions derived from caerin 1 peptides and some synthetic modifications. Molecular recognition initiating internal cyclisation of Glu23

The collision induced spectra of [M - H](-) anions from of caerin 1 peptides and some synthetic modifications show the usual alpha, beta and beta' backbone cleavages together with Ser (epsilon,gamma) and Glu (gamma) cleavages which break the peptide backbone in the vicinity of those residues. All of these cleavages require the peptide backbone to be flexible. There is also a backbone cleavage of a type not observed before. This cleavage involves nucleophilic attack of the carboxylate anion of the Glu23 side chain at the backbone CH of Ile 21. We propose that this cleavage requires the caerin peptide to be in an alpha helical conformation (the 3D structure that this peptide adopts in solution) in order that the interacting groups are held in close proximity.     

Enhanced ferroelectric phase content of polyvinylidene difluoride fibers with the addition of magnetic nanoparticles

Polyvinylidene difluoride (PVDF) fibers with continuously dispersed ferrite (Ni 0.5Zn 0.5Fe 2O 4) nanoparticles were prepared by electrospinning from dimethyl formamide (DMF) solutions. The effects of the electrospinning processing conditions and nanoparticle loading on the formation of the alpha, beta, and gamma phases of PVDF were studied using infrared spectroscopy and differential scanning calorimetry. The amount of the ferroelectric beta and gamma phases present in the fibers was found to increase with increased nanoparticle loading. We have shown that the formation of PVDF phases with extended chain conformations can be enhanced by the addition of a well-dispersed nanoparticle phase. At increased nanoparticle loadings, the alpha phase is completely converted to the more extended beta and gamma phases.