Haemoglobin Miyada,a β—δ Fusion Peptide (Anti-Lepore) Type discovered in a Japanese Family

SEVERAL slow-moving abnormal haemoglobins associated with thalassaemia-like stigmata have been encountered during the past 13 yr^1–6. The non-α-chains of these haemoglobins are the fusion products⁷ of δ and β-chains; the N-terminal end containing a part of the β-chain joined to a fragment of the β -chain ending in the C-terminal. But no structural variants of anti-Lepore type (fused in reverse β-δ) have yet been demonstrated. In a systematic screening survey for abnormal haemoglobins⁸, we discovered a new variant named Hb Miyada which may possess a fused non-α-chain.     

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.     

Organization of human δ- and β-globin genes in cellular DNA and the presence of intragenic inserts

We have analyzed human cellular DNA for its δ- and β-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with ³²P-β-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3′ end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the δ and β genes and those containing sequences flanking either 5′ to the δ gene or 3′ to the β gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, δ- and β-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.     

A comparison of the functional properties of two lepore hemoglobins with those of hemoglobin A1.

Lepore hemoglobins result from crossovers between normal beta and delta chain genes. Structural investigation of two newly discovered examples of Lepore hemoglobins revealed one of them to be structurally identical to hemoglobin Lepore Hollandia α₂^Aδ²² -x- β⁵⁰, a rarely occurring Lepore variant, while the second had the structure of hemoglobin Lepore Boston α₂^Aδ⁸⁷ -x- β¹¹⁶. Studies of the equilibrium and kinetic properties of the liganding reactions of these two Lepore hemoglobins, which differ only in three amino acid residues, and comparison of these with the known properties of hemoglobin A₁ (α₂β₂) and hemoglobin A₂ (α₂δ₂) have been carried out. A high value of n, the Hill coefficient, indicating normal heme-heme interaction, was observed in each hemoglobin along with a normal Bohr effect. However, a slight but definite increase in oxygen affinity was observed for each Lepore hemoglobin. Furthermore, kinetic studies indicated a slight but consistently increased rate of ligand combination and a somewhat decreased rate of oxygen dissociation for hemoglobins Lepore Hollandia and Lepore Boston at pH 7 and 20 °C. Apparently, the higher oxygen affinity of these Lepore hemoglobins over those of the normal hemoglobins A₁ and A₂ reflects changes of sequence that are common to both types of hemoglobin Lepore.     

Further identification of the nature and linkage of the carbohydrate in hemoglobin A1c.

We have found that Hb A_1c contains neutral sugars which are only partially hydrolyzed from the N-termini of β chains. In both normal and diabetic Hb A_1c, 0.2–0.3 equivalents of hexose were released, composed primarily of glucose and mannose in a 3:1 ratio. When Hb A_1c was reduced with ³HNa BH₄ and then treated with periodic acid, most of the radioactivity was recovered as ³H-formic acid with much lesser amounts of ³H-formaldehyde. From these results, we propose that in the red cell, glucose binds to the α-amino position of hemoglobin β-chains (valine) in an aldimine (Schiff base) linkage. This aldimine can then partially rearrange in a reversible manner to form a ketoamine linkage which is stable to acid hydrolysis. This Amadori-type rearrangement accounts for the formation of mannose, the C-2 epimer of glucose, as well as the inability to demonstrate ³HNa BH₄ reduction at the C-1 position.     

Primary structure of the hemolglobin β-chain of Rose-ringed Parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin β-chain was established, completing the analysis of this hemoglobin. Comparisons with other avian β-chains show variations smaller than those for the corresponding α-chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform β-chain, and a total of 35 positions are affected by differences among all avian β-chains analyzed (versus 61 for the α-chains). At three positions, the Psittacula β-chain has residues unique to this species. Three α₁β₁ contacts are modified, by substitutions at positions β51, β116, and β125.     

Electrophoretic and chromatographic evidence for allelic polymorphisms in the river buffalo α-globin gene complex

Isoelectric focusing in the ultranarrow immobilized (7.1–7.5) pH gradient (IPG) of hemoglobin and high-performances liquid chromatography (HPLC) of globin chains were used to investigate Hb polymorphism in Italian river buffalo. Six different phenotypes, each characterized by two or four different Hbs, were detected by IPG, whereas two different^IIα-globin chains were separated from two different^Iα-chains by HPLC. Two α-chains (^Iα¹ and^IIα³), and Hbs with similar mobilities (Hb1 andHb3), were associated with the AA Hb phenotype: two α-chains (^Iα² and^IIα⁴), and Hbs with different mobilities (Hb2 andHb4), were associated with the BB phenotype: two sets of doublet Hbs were associated with the AB phenotype, thus suggesting allelic polymorphisms at the two α loci. An allele at the β locus is responsible for increasing to as many as eight the number of different Hbs, thus further complicating the notable Hb polymorphism of the river buffalo.     

Separation of β22 dimer from bovine bone collagen

The precise role of the α₂-chain in collagen type I is of considerable scientific interest. Our recent studies demonstrated that the most noticeable difference between type I collagens, which were obtained from bovine hard tissues (bone, dentine) and soft tissues (tendon, skin), was presented in the position of β chain dimers using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The additional band observed both in the bone and dentine collagen was putatively identified as β₂₂ dimer (made of by an intermolecular cross-linking between two α₂-chains). Further investigations carried out on bovine bone and skin collagen, corresponding to hard tissue and soft tissue collagen respectively, confirmed this hypothesis. Successful separation of individual β₂₂ dimer from bone collagen was achieved. The procedure involves molecular-sieve chromatography on a Sephacryl S-400 column followed by differential acetone precipitation. Identification was done by the widely used methods, such as SDS-PAGE and cyanogen bromide (CNBr)-cleaved peptide analysis. It was proposed that the dimer and consequently α₂-chains may play important roles in the morphological and biological differences between hard and soft tissues.     

GABAA Receptors of Cerebellar Granule Cells in Culture: Interaction with Benzodiazepines

GABA_A receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABA_A receptors is the presence in cerebellar granule cells of subunits such as α6 and δ which give insensitivity to classical benzodiazepines. In fact, cerebellar GABA_A receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive α1 β2/3 γ2 receptors from those which are diazepam-insensitive and contain the α6 subunit. This is achieved by utilizing furosemide/Zn²⁺ which block α6 containing and incomplete receptors.     

Physical properties of type i collagen in solution: Structure of α-Chains and β- and γ-components and two-component mixtures

The structure of thermally denatured Type I collagen has been studied using laser light scattering. The results indicate that the diffusion coefficients of α-chains and β- and γ-components are 1.550 ± 0.08 × 10^?7, 1.000 ± 0.05 × 10^?7, and 0.835 ± 0.04 × 10^?7 cm²/sec, respectively, at temperatures between 20 and 40°C. It is concluded from diffusion data that these species have hydrodynamic radii of about 13.8 nm (α-chain), 21.5 nm (β-component), and 25.7 nm (γ-component), consistent with previous studies of thermal denaturation by light scattering. It is also concluded, based on volume calculations, that a large volume increase occurs when the triple helix unfolds. Homodyne correlation functions for two component mixtures of α-chains and β-and γ-components appeared to decay exponentially. In all but one case discussed the correlation function could be fitted with a single component having a translational diffusion coefficient which was an intensity weighted average of the diffusion coefficient of each component present.     

Amino acid differences between the α-chains from two hemoglobins of the yellow-cheeked vole (family cricetidae)

The yellow-cheeked vole (Microtus xanthognathus) shows two electrophoretic hemoglobin components. Electrophoresis of the polypeptide chains from the separated hemoglobin components shows identical β-chains but two α-chains of different mobility, α ^f and α ^s . The composition of soluble tryptic peptides was determined for each α-chain. Amino acid differences were found in peptides αT1 and αT9; the compositions of the remainder of the homologous peptides were identical. Differences in αT1, found at α4 (α ^s -Gly-α ^f -Val) and α5 (α ^s -Thr-α ^f -Asp), were confirmed after a run to residue 20 of the fast component in an automatic sequencer. The differences in charge between αT1 peptides can account for the electrophoretic pattern of two hemoglobins. This is the first time that it has been possible to identity the residues which can account for the charge difference between the two hemoglobins observed in a Microtus species.     

Duplicated α-Chain Genes in Hopkins-2 Haemoglobin of Man and Evidence for Unequal Crossing Over between Them

SYNTHESIS of the α and β-chains for haemoglobin is dictated by independent genetic loci. The first evidence for this notion came from Smith and Torbert's observation that inheritance of haemoglobin Hopkins-2 (Ho-2) was independent from that of haemoglobin S¹ and that Ho-2 was an α-chain variant². We wish to report the amino-acid replacements involved. These structural changes establish the presence of at least two α-chain genes in man. Some physical and physiological properties of the abnormal haemoglobin and the clinical status of carriers, have been reported in another article³.     

Fragmentation of chloroplast coupling factor in dependence of bound nucleotides Preparation of a reconstitutionally active form of subunit δ

Previous studies on the ability of CF₁, fragments to reconstitute photophosphorylation in CF₁,-depleted thylakoids have shown that the degree of reconstitution was correlated with the presence of subunit δ in the fragment. This was taken as evidence that subunit δ was necessary for plugging the active proton channel CF₀ [(1986) Eur. J. Biochem. 160, 635–643]. We questioned whether or not δ alone had this ability. In order to obtain δ we investigated the role of bound nucleotides in the stability of CF₁. Starting from ammonium sulfate-precipitated CF₁, we found that a low content of bound ADP (1 mol ADP/mol CF₁) seemed to stabilize the β—δ interaction, while loosening the interaction between α,β and γ. By elution from an anion-exchange column in the presence of the nonionic surfactant Mega 9 we obtained β₃δ and CF₁(—δ) (both containing one ADP) or, after washing with alcohol/glycerol mixtures, β (nucleotide-free) and CF₁/CF₁(—ϵ). On the other hand, with a further 2 ADP and 2 ATP bound to CF₁, (after incubation with excess ATP) the α-β-γ interaction was stabilized in such a way that subunit δ alone could be isolated from the complex. Subunit δ, when isolated by this procedure and added back to CF₁-depleted thylakoids, reconstituted a high rate of photophosphorylation.     

Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H₂O₂), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS² mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the β-globin chain of the Hb:Hp complex, including decreased βCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein.     

Subunit equivalence in Escherichia coli and bovine heart mitochondrial F1F0 ATPases

F₁-ATPases from bovine mitochondria and Escherichia coli both contain 5 subunits named α, β, γ, δ and ε. Sequence analysis shows that the δ subunits are not related, nor are the ε subunits. The counterpart of mitochondrial δ is bacterial ε. The subunit equivalent to bacterial δ is mitochondrial oligomycin sensitivity conferral protein.     

Structure of α6β3δ GABAA receptors and their lack of ethanol sensitivity

Delta (δ) subunit containing GABA_A receptors are expressed extra‐synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with α₆ subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA_A receptor pentamers by subunit concatenation. These receptors (composed of α₆, β₃ and δ subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one and to 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. α₆‐β₃‐α₆/δ receptors showed a substantial response to GABA alone. Three receptors, β₃‐α₆‐δ/α₆‐β₃, α₆‐β₃‐α₆/β₃‐δ and β₃‐δ‐β₃/α₆‐β₃, were only uncovered in the combined presence of the neurosteroid 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one with GABA. All four receptors were activated by 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the δ subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the δ subunit can assume multiple positions in a receptor pentamer composed of α₆, β₃ and δ subunits.     

Identification of covalent binding sites of ethyl 2-cyanoacrylate,methyl methacrylate and 2-hydroxyethyl methacrylate in human hemoglobin using LC/MS/MS techniques

Acrylates are used in vast quantities, for instance in paints, adhesive glues, molding. They are potent contact allergens and known to cause respiratory hypersensitivity and asthma. Here we study ethyl 2-cyanoacrylate (ECA), methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA). There are only limited possibilities to measure the exposure to acrylates, especially for biological monitoring. The aim of the present study was to investigate the chemical structures of adducts formed after reaction of hemoglobin (Hb) with ECA, MMA, and HEMA. This information may be used to identify adducted Hb peptides for biological monitoring of exposure to acrylates. Hb-conjugates with ECA, MMA, and HEMA were synthesized in vitro. The conjugates were digested by trypsin and pronase E. Adducted peptides were characterized and analyzed by liquid chromatography and nano electro spray/hybrid quadrupole time-of-flight mass spectrometry (MS) as well as tandem quadrupole MS. The search for the adducted peptides was facilitated by visualizing the MS data by different computer programs. The results showed that ECA binds covalently to cysteines at the 104 position in the α and the position 112 in the β-chains in Hb. MMA and HEMA bound to all the cysteines in both chains, Cys¹⁰⁴ in the α-chain and Cys⁹³ and 112 in the β-chain. The full-length spectra of in un-digested Hb confirmed this binding pattern. There was no reaction with N-acetyl-l-lysine at physiological pH. The adducted peptides were possible to measure using LC/MS/MS in selected reaction monitoring mode. These peptides may be used for biological monitoring of exposure to ECA, MMA and HEMA.