Oxygen equilibria of hemoglobin A and hemoglobin S valency hybrids.

Oxygen equilibrium determinations with “unsymmetrical” MetHb/Hb hybrids derived from human hemoglobins A and S are reported. All four of the possible hybrids have higher oxygen affinity than the parent hemoglobins. The α₂^Metβ₂^S hybrid has a lower oxygen affinity than that of α₂^Metβ₂^S. However, both the β^Met hybrids have similar oxygen affinity. The Bohr value of α₂^Metβ₂^S is more negative than that of α₂^Metβ₂^A while the β^Met hybrids appear to have almost identical Bohr values. These findings favor the view that α and β chains in hemoglobin A have different conformations and indicate that hemoglobin S has a β-chain conformation different from that of β-chain of hemoglobin A. This difference is probably carried into the oxygenation properties of the α-chain in such a way as to be reflected only when the β chain is oxidized.     

Observation of allosteric transition in hemoglobin

Two conclusions have been drawn from NMR studies of mixed state hemoglobins. First the α and β subunits in hemoglobin are not equivalent in their conformational properties. Second the mixed state hemoglobin (α^IIICN β^II)₂ can take two different quaternary structures without changing the degree of ligation. One of the two structures is similar to that of deoxyhemoglobin and the other to that of oxyhemoglobin.     

A dimeric polyoxometalate sandwich motif containing a truncated {Mn3O4} cubane core

A novel sandwich-type silicotungstate motif, K₁₈[Mn^II₂{Mn^II(H₂O)₅Mn^III₃(H₂O)(B-β-SiW₉O₃₄)(B-β-SiW₆O₂₆)}₂]·20H₂O 1, has been isolated from the reaction of K₈[γ-SiW₁₀O₃₆]·12H₂O and manganese ions in aqueous acidic media. The transition metal-substituted polyoxometalate (TMSP) 1 has been fully characterized by single-crystal X-ray diffraction, elemental analysis, thermogravimetric analysis, and infrared spectroscopy. This dimeric polyanion consists structurally of the sandwich polyanion {Mn^II(H₂O)₅Mn^III₃(H₂O)(B-β-SiW₉O₃₄)(B-β-SiW₆O₂₆)}, dimerized via two manganese(II) linker ions. Each monomeric unit is composed of two non-equivalent Keggin fragments, (B-β-SiW₈O₃₁) and (B-β-SiW₆O₂₆), linked to each other via three manganese ions resulting in a truncated {Mn₃O₄} cubane core. Experimental, structural, and electrochemical aspects of the material are reported and discussed.     

Conformational requirements for the polymerization of hemoglobin S: studies of mixed liganded hybrids

Gelation experiments with artificially formed half-liganded hybrid tetramers of hemoglobin S demonstrate that when either the α chains or the β^s chains are fixed in the cyanmet (CNmet) liganded state, gelation occurs upon deoxygenation of the ferrous chains. The minimum concentration of hemoglobin required for gelation is equivalent for both hybrids (α₂^cnmetβ₂^s and α₂β₂^scnmet), is considerably higher than the concentration required to gel deoxy-Hb S (α₂β₂^s), and can be restored to the lower minimum gelling point of α₂β₂^s by reduction of the CNmet chains with dithionite. These results suggest that the most important conformational determinant of the deoxy state for polymerization of Hb S is the quaternary deoxy structure rather than the tertiary structural effect of the ligand state of the α or the β^s chains, and are furthermore consistent with the notion that asymmetric deoxy-CNmet hybrid tetramers assume a conformation which resembles, but is not identical to that of deoxyhemoglobin.The results of gelation experiments with mixtures of hemoglobins S and A in which selected chains of one or both hemoglobins are in the CNmet form support the concept that certain non-S hemoglobins may participate in the sickling process by forming hybrid tetramers with Hb S (such as α₂β^aβ^s). The conformational requirement for participation of these hybrids in polymers also appears to be a quaternary deoxy-like structure.     

Studies on cobalt myoglobins and hemoglobins, XIII. A consequence of the occurrence of glutamine at the E7 (58) site of alpha subunits in opossum hemoglobin

To investigate the mode of interactions between heme metal, bound oxygen and the distal residue at the E7 site, we have measured accurate oxygen equilibrium curves, oxygen binding relaxations following temperature-jump, and electron paramagnetic resonance spectra of natural and cobalt-substituted opossum hemoglobin, which has glutamine and histidine at the E7 site of the α chain and the β chain, respectively, and compared them with those of natural and cobalt-substituted human hemoglobin, which has histidine at the E7 site of both the α and β chains.Natural opossum hemoglobin has a lower oxygen affinity, slightly smaller and pH-dependent co-operativity, a somewhat greater Bohr effect, and a smaller effect of organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate on oxygen affinity as compared to natural human hemoglobin. Upon substitution of cobalt for iron, these oxygenation characteristics of opossum hemoglobin relative to those of human hemoglobin were preserved well. The behavior of the intrinsic oxygen association constants pertaining to the four oxygenation steps (i.e. the Adair constants) upon addition of the organic phosphates or pH changes indicates that the allosteric equilibrium in opossum hemoglobin is biased towards the T state as compared with that in human hemoglobin, and that the oxygen affinity of the R structure is lower for opossum hemoglobin than for human hemoglobin. The temperature-jump kinetic data indicate that the lower oxygen affinity of opossum cobalt-hemoglobin in comparison with that of human cobalt-hemoglobin can be ascribed to a decreased oxygen association rate constant. The electron paramagnetic resonance experiments on oxy and deoxy opossum and human cobalt-hemoglobins in buffered H₂O and ²H₂O, including their photolysed products at a low temperature, provided the following information. The cobaltous ion of the α subunits of deoxy opossum cobalt-hemoglobin is in an environment that is similar to that for cobaltous ions of deoxy human cobalt-hemoglobin in the T state. The hydrogen bond between the bound oxygen and the residue at E7, which has been shown to exist in oxy human cobalt-hemoglobin and oxy sperm whale cobalt-myoglobin, is absent or, at least, significantly altered in the α subunits of oxy opossum cobalt-hemoglobin, probably resulting in a lower oxygen affinity. Interference by isoleucine at E11α with an oxygen molecule is suggested as an explanation for the lowered affinity of opossum iron-hemoglobin. However, no straightforward structural explanation is available for the lower oxygen affinity of the R structure and the allosteric equilibrium biased towards the T state in opossum iron-hemoglobin.     

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.     

Quaternary structures and low frequency molecular vibrations of haems of deoxy and oxyhaemoglobin studied by resonance Raman scattering

The influence of quaternary structure on the low frequency molecular vibrations of the haem within deoxyhaemoglobin (deoxy Hb) and Oxyhaemoglobin (oxy Hb) was studied by resonance Raman scattering. The FeO₂ stretching frequency was essentially identical between the high affinity (R) state (Hb A) and low affinity (T) state (Hb Kansas and Hb M Milwaukee with inositol hexaphosphate). However in deoxy Hb, only one of the polarized lines showed an appreciable frequency shift upon switch of quaternary structure, i.e. 215 to 218 cm^?1 for the T state (Hb A, des-His(146β) Hb, and des-Arg(141α) Hb (pH 6.5)) and 220 to 221 cm^?1 for the R state (des-Arg(141α) Hb (pH 9.0), des-His(146β)-Arg(141α) Hb and NES des-Arg(141α) Hb). Based on the observed ⁵⁴Fe isotopic frequency shift of the corresponding Raman lines of deoxy Hb A (214 → 217 cm^?1), of deoxy NES des-Arg Hb (220 → 223 cm^?1), of the protoporphyrinato-Fe(II)-(2-methylimidazole) complex in the ferrous high spin state (207 → 211 cm^?1) and of deoxymyoglobin (220 → 222 cm^?1) (Kitagawa et al., 1979), and on substitution of perdeuterated for protonated 2-methylimidazole in the deoxygenated picket fence complex (T_pivPP)Fe²⁺ (2-MeIm) (209 → 206 cm^?1), and on the results of normal co-ordinates calculation carried out previously, we proposed that the 216 cm^?1 line of deoxy Hb is associated primarily with the FeN_ε(HisF8) stretching mode and accordingly that the FeN_ε(HisF8) bond is stretched in the T state due to a strain exerted by globin.     

Spin-state selection filters for the measurement of heteronuclear one-bond coupling constants

Novel α/β-half-filter elements are proposed for the separation of the high-field and low-field component of ¹ J _HC and ¹ J _HN splittings into different subspectra. The α/β-half-filter elements are of the same duration as the S³CT pulse sequence element and, like this, are less sensitive to cross talk between different subspectra than the original shorter α/β-half-filters. The filter elements are demonstrated with the measurement of ¹ J _HC coupling constants of C^αH groups in 2D and 3D experiments and the subspectral editing of the four different multiplet components observed in two-dimensional α/β-HSQC-α/β spectra recorded without heteronuclear decoupling in either dimension.     

Synthesis and structures of dimeric iron(III)-Oxo and -imido complexes containing intramolecular hydrogen bonds

Hydrogen bonding networks proximal to metal centers are emerging as a viable means for controlling secondary coordination spheres. This has led to the regulation of reactivity and isolation of complexes with new structural motifs. We have used the tridenate ligand bis[(N′-tert-butylureido)-N-ethyl]-N-methylaminato ([H₂1]²⁻) that contains two hydrogen bond donors to examine the oxidation of the Fe^II-acetate complex, [Fe^IIH₂1(η²-OAc)]⁻ with dioxygen, amine N-oxides, and xylyl azide. A complex with Fe^III-O-Fe^III core results from the oxidation with dioxygen and amine N-oxides, in which the oxo ligand is involved in hydrogen bonding to the [H₂1]²⁻ ligand. A distinctly different hydrogen bonding network was found in Fe^III dimer isolated from the reaction with the xylyl azide: a rare Fe^III-N(R)-Fe^III core was observed that does not have hydrogen bonds to the bridging nitrogen atom. The intramolecular H-bond networks within these dimers appear to adjust to the presence of the bridging species and rearrange to its size and electron density.     

Relationship between β4 hydrogen bond and β6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S

Oversaturated deoxy-α₂β₂^T4V aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-α₂β₂^E6V,D73H. Solubility of deoxy-α₂β₂^T4V was ∼10-fold lower than that of deoxy-HbS and was similar to oxy- and deoxy-α₂β₂^E6V,T4V. These results indicate that β4Val in HbA in the oxy and deoxy forms with or without β6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a β4/β73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(α₂β₂^E6V,D73N), while α₂β₂^T4V and α₂β₂^D73H as well as HbS, α₂β₂^E6V,D73H and α₂β₂^E6V,T4V in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of β6 amino acid hydrophobicity that the synergism between the β4Thr hydrogen bond and β6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition.     

The effects of solvent on dioxygen binding to cobalt(II) picket fence porphyrins

The O₂ affinities on base adducts of four atropisomers of picket fence porphyrin Co(TpivPP), and the corresponding α⁴ complex containing valeramido pickets instead of pivalamido Co(α⁴-TvalPP) were measured in several solvents at O or −15 °C. The O₂ affinities of the α⁴ complexes are the lowest in DMF which is the most polar of the solvents used, while those of the other isomers are the highest in DMF. This observation was explained in terms of direct and indirect interactions between the solvent and the bound O₂. The trans-α² complex shows higher O₂ affinity in dichloromethane than those in aromatic solvents because of the preferential solvation of the deoxy complex in the solvents. The variation of the O₂ affinities of this system to solvents is considerably smaller than those of ‘flat porphyrin’ complexes. This result suggested that the pocket polarity introduced by the amide groups weakens the solvent–solute interaction on the O₂ affinities of this system and also that the solvation of the oxy state rather than the deoxy state predominantly affects the O₂ affinities. It was concluded that the enhanced O₂ uptake by the picket fence may be due to the stabilization of the oxy state by intramolecular interactions rather than to destabilization of the deoxy state by inhibiting solvation for the active site.     

Assignment of proximal histidyl imidazole exchangeable proton NMR resonances to individual subunits in hemoglobins A,Boston, Iwate and Milwaukee

The proton nmr spectra of the synthetic valency hybrids, α₂(β⁺CN)₂, (α⁺CN)₂β₂ of hemoglobin A and the natural valency hybrids of the mutant hemoglobins Boston, Iwate and Milwaukee have led to the unambiguous assignment of the two proximal histidyl imidazole exchangeable proton signals at 64 and 76 ppm to individual α and β subunits, respectively. New single non-exchangeable proton resonances detected in the extreme downfield region of the spectra of Hbs Boston and Iwate are tentatively assigned to the coordinated tyrosine of the mutated α chains.     

Nitric oxide binding to cationic and anionic ferric porphyrins in aqueous solution: reversible formation of ferrous NO species of the cationic porphyrin

Two water-soluble ferric porphyrins, sodium 5α,10β,15α,20β-tetrakis(2-(sulfonatoacetamido)phenyl)porphyrinatoiron(III) (Fe^IIITanP) and 5α,10β,15α,20β-tetrakis(2-(N,N,N-trimethylammoniumacetamido)phenyl)porphyrinatoiron(III) chloride (Fe^IIITcatP), were synthesized. The pK_a values of the coordinated H₂O of Fe^IIITanP and Fe^IIITcatP were evaluated to be 8.0 and 4.1, respectively. Reactions of NO with the ferric porphyrins were examined spectrophotometrically in aqueous solution. Porphyrin Fe^IIITanP binds NO reversibly to give the corresponding ferric NO species at pH 1.3 and pH 3.0, and Fe^IIITcatP reacts similarly with NO at pH 1.3. The thermodynamic data for the NO binding were estimated from van't Hoff plots. At pH 3.0, visible and ESR spectral data indicated that Fe^IIITcatP binds NO reversibly to produce ferrous NO species depending on NO partial pressures. These results were discussed based on through-space intramolecular interactions between the coordinated H₂O or NO and the ionic substituents of the porphyrins.     

Hb Natal or α2(minus Tyr-Arg)β2: A high oxygen affinity α chain variant with a deleted carboxy-terminus resulting from a TAC → TAA (Tyr → terminating codon) mutation in codon α140

The discovery is reported of a fast-moving α chain variant (Hb Natal) which is characterized by a shortened α polypeptide chain because of the deletion of the Tyr-Arg carboxy-terminal residues. Through amplification of appropriate segments of DNA and hybridization with synthetic oligonucleotide probes, it was possible to detect a C → A mutation in codon 140 of the α₂ globin gene, which causes a change in the codon for tyrosine to a terminating codon. Hb Natal or α₂(minus Tyr-Arg)β₂ has a high affinity for oxygen without a Bohr effect and heme-heme interaction. These results provide direct evidence for the importance of the tyrosine residue at α140 in the oxygenation-deoxygenation process.     

Structure of deoxyhaemoglobin Yakima: a high-affinity mutant form exhibiting oxy-like 1 2 subunit interactions

Crystals of deoxyhaemoglobin Yakima (Asp Gl(99)β → His) are isomorphous with those of deoxyhaemoglobin A, even though the mutation produces disturbances in both the tertiary structure of the subunits and the quaternary structure of the tetramer. Asp Gl(99)β₂ lies at the α₁β₂ subunit interface, and in deoxyhaemoglobin A forms a crucial hydrogen bond with Tyr C7(42) α₁. The histidine residue that replaces the aspartate results in the removal of this single important intersubunit bond, and it further acts as a wedge between the α₁ and β₂ subunits, so that they are pushed apart and displaced part of the way towards the oxy structure. These disturbances are accompanied by the formation of a new intersubunit hydrogen bond, which is usually only observed in the oxy quaternary structure of haemoglobin. The disturbances at the α₁β₂ contact affect the stereochemistry of the entire molecule and are transmitted to the α and β haems. The X-ray structure of deoxy Yakima therefore provides a stereochemical explanation for its abnormal function; this being an abnormally high affinity for oxygen and vastly diminished haem-haem interactions.     

Covalent anthocyanin–flavonol complexes from the violet-blue flowers of Allium ‘Blue Perfume’

Three covalent anthocyanin–flavonol complexes (pigments 1–3) were extracted from the violet-blue flower of Allium ‘Blue Perfume’ with 5% acetic acid-MeOH solution, in which pigment 1 was the dominant pigment. These three pigments are based on delphinidin 3-glucoside as their deacylanthocyanin and were acylated with malonyl kaempferol 3-sophoroside-7-glucosiduronic acid or malonyl-kaempferol 3-p-coumaroyl-tetraglycoside-7-glucosiduronic acid in addition to acylation with acetic acid.By spectroscopic and chemical methods, the structures of these three pigments 1–3 were determined to be: pigment 1, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-(3^III-O-(β-glucopyranosyl^V)-β-glucopyranosyl^III)-4^II-O-(trans-p-coumaroyl)-6^II-O-(β-glucopyranosyl^IV)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))) malonate; pigment 2, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-β-glucopyranosyl^III)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))); and pigment 3, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-(3^III-O-(β-glucopyranosyl^V)-β-glucopyranosyl^III)-4^II-O-(cis-p-coumaroyl)-6^II-O-(β-glucopyranosyl^IV)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))) malonate.The structure of pigment 2 was analogous to that of a covalent anthocyanin–flavonol complex isolated from Allium schoenoprasum where delphinidin was observed in place of cyanidin. The three covalent anthocyanin–flavonol complexes (pigment 1–3) had a stable violet-blue color with three characteristic absorption maxima at 540, 547 and 618 nm in pH 5–6 buffer solution. From circular dichroism measurement of pigment 1 in the pH 6.0 buffer solution, cotton effects were observed at 533 (+), 604 (−) and 638 (−) nm. Based on these results, these covalent anthocyanin–flavonol complexes were presumed to maintain a stable intramolecular association between delphinidin and kaempferol units closely related to that observed between anthocyanin and hydroxycinnamic acid residues in polyacylated anthocyanins. Additionally, an acylated kaempferol glycoside (pigment 4) was isolated from the same flower extract, and its structure was determined to be kaempferol 3-O-sophoroside-7-O-(3-O-(malonyl)-β-glucopyranosiduronic acid).     

The kinetics of ligand substitution in bis(N-alkylsalicylaldiminato)nickel(II) Complexes: a study on the reactivity of square-planar versus octahedral coordination

The bis(N-alkylsalicylaldiminato)nickel(II) complexes Ni(R-sal)₂ with R = CH(CH₂OH)CH(OH)Ph (I), R = CH(CH₃)CH(OH)Ph (II) and R = CH₂CH₂Ph (III; Ph = phenyl) were prepared and characterized. In the solid state I and II are paramagnetic (μ = 3.2 and 3.3 BM at 20 °C, respectively), whereas III is diamagnetic. It follows from the UV-Vis spectra that in acetone solution I is six-coordinate octahedral and III is four-coordinate planar, the spectrum of II showing characteristics of both modes of coordination. Vis spectrophotometry and stopped-flow spectrophotometry were applied to study the kinetics of ligand substitution in I–III by H₂salen (= N,N′-disalicylidene-ethylenediamine) in the solvent acetone at different temperatures. The kinetics follow a second-order rate law, rate = k[H₂-salen] [complex]. At 20 °C the sequence of rate constants is k(III):k(II):k(I) = 11 850:40.6:1. The activation parameters are ΔH^≠(I) = 112, ΔH^≠(II) = 40.7, ΔH^≠(III) = 35.7 kJ mol⁻¹ and ΔS^≠(I) = 92, ΔS^≠(II) = −103, ΔS^≠(III) = −89 J K⁻¹ mol⁻¹. The enormous difference in rate between complexes I, II and III, which is less pronounced in methanol, is attributed to the existence of a fast equilibrium planar ⇌ octahedral, which is established in the case of I and II by intramolecular octahedral coordination through the hydroxyl groups present in the organic group R. An A-mechanism is suggested to control the substitution in the sense that the entering ligand attacks the four-coordinate planar complex, the octahedral complex being kinetically inert.     

Subunit component and their roles in the sodium-transport NADH: quinone reductase of a marine bacterium,Vibrio alginolyticus

The sodium-transport respiratory chain NADH: quinone reductase of a marine bacterium, Vibrio alginolyticus, was purified by high-performance liquid chromatography. The purified quinone reductase, which catalyses the reduction of ubiquinone to ubiquinol, was composed of three subunits, α, β and γ, with apparent molecular weights of 52 000, 46 000 and 32 000, respectively. The subunit β contained one molecule of FAD per molecule and catalysed the reduction of ubiquinone to ubisemiquinone. The subunit α contained FMN as a prosthetic group. The quinone reductase was reconstituted from α and βγ, but not from α and β, and the maximum activity was obtained at the equimolar amounts of FAD(β) and FMN(α). The molecular weight of quinone reductase complex was estimated to be 254 000, which corresponded to a dimer of αβγ complex or α₂β₂γ₂. The subunit γ increased the affinity of β for ubiquinone-1. The reaction catalysed by FMN-containing α-subunit was essential for the generation of membrane potential in proteoliposomes and the coupling site of sodium pump in the quinone reductase was localised to this reaction step.     

Synthese der “repeating unit” der O-spezifischen kette des lipopolysaccharides aus Aeromonas salmonicida

8-Methoxycarbonyloctyl 2-azido-4,6-O-benzylidene-2-deoxy-β-d-mannopyranoside reacted with 2,3,4-tri-O-acetyl-α-l-rhamnopyranosyl bromide to give a disaccharide from the which the glycosyl-acceptor 8-methoxycarbonyloctyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2,4,-di-O-acetyl-α-l-rhamnopyranosyl)-β-d-manno pyranoside (19) was obtained. This glycosyl-acceptor with 2,3,4,6-tetra-O-benzyl-α-d-glucopyranosyl chloride to give trisaccharide derivative 22 and with 2,3,6-tri-O-(α-²H₂)benzyl-4-O-(2,3,4,6-tetra-O-(α-²H₂)benzyl-α-d-glucopyranosyl)-α-d-glucopyranosyl chloride to give tetrasaccharide derivative 29. Deblocking of 22 yielded 8-methoxycarbonyloctyl O-(α-d-glucopyranosyl)-(1→3)-O-α-l-rhamnopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-mannopyranoside and deblocking of 29 8-methoxycarbonyloctyle O-α-d-glucopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→3)-O-α-l-rhamnopyranosyl- (1→3)-2-acetamido-2-deoxy-β-d-mannopyranoside. Both oligosaccharides represent the “repeating unit” of the O-specific chain of the lipopolysaccharide from Aeromonas salmonicida.