Reversible oxygenation of heme bound to polyvinylpyridine and polyvinylimidazole

The interaction between heme bound to poly-4-vinylpyridine (PVP) or poly-N-vinyl-2-methylimidazole (PVMI) and molecular oxygen (O₂) was studied. In this paper, the reactions of some types of heme with O₂ in organic solvents, particularly in N,N-dimethylformamide (DMF) were discussed. The free heme not bound to an axial base was easily oxidized irreversibly to hemin in DMF with bubbled O₂. The hemochromogens complexed with pyridine, imidazole, or their polymeric derivatives such as PVP and PVMI bound O₂ to one of the axial coordination sites. The characteristic absorption band assignable to the resulting oxygenated heme was observed at 402 nm. This absorption band could be changed back to the characteristic band of the reduced hemochromogen at 418 nm by removing O₂ dissolved in the DMF solution by a vacuum or by a stream of nitrogen. Thus, the hemochromogens bound to the synthetic polymers were found to adsorb and desorb O₂ reversible in DMF. When the polymeric ligands were used, the equilibrium constants in the complexation of heme with these polymers were about 10² times as large as those of the corresponding monomeric ligands. The oxygenation rates and the capacities of O₂ of the polymeric hemochromogens were larger than those of the monomeric hemochromogens. In addition, the oxygenation rate of the polymer complex was changeable owing to the conformational change of the polymeric ligand; this rate increased about ten times under the optimal condition.     

Potentiometric titration of poly-S-carboxymethyl-L-cysteine in aqueous NaCl solution

pH titration measurements of poly- S-carboxymethyl-L -cysteine were undertaken in the aqueous Nacl solution in relation to the β form–random coil transition. The titration curves show a marked molecular weight dependence because of the shortened chain length of materials. Comparison of the optical rotatory dispersion parameter a₀ with the titration curve reveals that the titration curve apparently reflects a β structure–random coil transition. The β form of this polymer is assumed to be an intramolecular β form, rather than a β structure stabilized by an intermolecular hydrogen bond, at least in the polymer concentration range considered here. The standard free energy change per amino acid residue for the transition from un-ionized random coil to un-ionized β form is estimated to be about ?750 cal/mole residue in the range of 0.005–0.2M NaCl concentration.     

Molecular oxygen binding with α and β subunits within the R quaternary state of human hemoglobin in solutions and porous sol–gel matrices

Nanosecond laser flash-photolysis technique was used to study bimolecular and geminate molecular oxygen (O₂) rebinding to α and β subunits within oxygenated human adult hemoglobin in solutions and porous wet sol–gel matrices. Plasticity associated with the tertiary structure within R-state hemoglobin is explored through measurements that focus on the functional properties of hemoglobin under conditions designed to tune the tertiary structure without inducing the R to T transition. Inequivalence in the O₂ binding to the α and β hemes within the R quaternary structure is studied. The individual kinetic properties of the α and β subunits within the hemoglobin encapsulated in sol–gels and aged as the oxy derivative are shown to be independent of proton concentration over the pH range from 6.3 to 8.5. However, buffer effects on the subunits' properties are revealed in sol–gel-free mediums. Interestingly, the α and β subunits within the encapsulated hemoglobin possess the O₂ rebinding properties which fall within the range of the ones for oxygenated hemoglobin in the buffer solutions. The combined results show a pattern in which there is a progression of functional properties that are ascribed to a family of conformational substates of R-state hemoglobin. O₂ rebinding to the α and β subunits within the oxygenated R-state hemoglobin in both solutions and wet sol–gels is revealed to be modulated by tertiary structural changes in two quite different ways. The possible structural changes, which modify the O₂ rebinding properties, are discussed.     

Calculations of the Conformations of Iturin A in Relation with NMR Studies

Abstract

Iturin A is an antifungal antibiotic which was isolated from a strain of Bacillus subtilis, and contains a lipophilic β amino acid closing an heptapeptide cycle with polar L and D residues. Iturin A belongs to a lipopeptide family of which the LDDLLDL sequence is kept constant.

NMR spectroscopy and semi-empirical energy calculations are combined to design the conformations of Iturin A in pyridine solution. J coupling constants and nOes (nuclear Overhauser enhancements) are used as guiding line for energy calculations. This preliminary study shows that Iturin A in pyridine appears as rather rigid, especially in the L Pro 5—D Asn 6 region, probably involved in a β turn. The polar side chains can form different networks of intramolecular hydrogen bonds. The Tyr side chain, relatively mobile, could be involved in interactions with an hydrophobic environment as the β amino acid side chain found away from the peptide cycle.     

Synthèse et structure de la poly-O-carbobenzoxy L- tyrosine et de copolymères de O-carbobenzoxy L-tyrosine et de glutamate de benzyle ou d'aspartate de benzyle

The optical rotatory dispersion of copolymers of O-carbobenzoxy-L -tyrosine and benzyl L (or D )-glutamate as well as benzyl L -aspartate, dissolved in nonpolar solvent, has been studied. Moffitt's equation permits the determination of b₀ coefficients whose variation, with varying composition in amino acid residues, suggests that the molecules of poly-O-carbobenzoxy-L -tyrosine have a helical structure similar to that of poly-(benzyl L -glutamate). Results obtained from infrared spectroscopy and x-ray diffraction show that the copolymers possess a helical conformation in the solid state, even when they are very rich in carbobenzoxy-L -tyrosine residues. The value of the b₀, coefficient for poly-O-carbobenzoxy-L -tyrosine may be explained by a regular stacking of the chromophore groups around the helical backbone. The ordering of the molecules of this polymer in a purely helical structure seems favored by the insertion of a small number of foreign residues in the polypeptide chain.     

Oxygen activation and defence against oxygen toxicity in a psychrophilic bacteroidaceae

When suddenly exposed to air the growth of the obligate anaerobic bacterium of the bacteroidaceae type, strain B6, continues for a few hours before coming to a complete stop. When air is shut off soon after growth has ceased, the organism is able to reestablish anaerobic conditions due to an ability to reduce O₂, and resumes normal growth after another few hours. The O₂ reducing ability of the organism is due to the presence in the cells of a particlebound NADH oxidase, a soluble NADPH oxidase and a soluble pyruvate oxidase. The two pyridine nucleotide oxidase reduce O₂ to H₂O₂, the pyruvate oxidase reduces O₂ to H₂O. Catalase and peroxidase were not detected in anaerobically grown cells. Kinetic studies with cell-free extracts showed that the pyruvate oxidase had a considerably greater affinity (smaller K _m) for O₂ and capacity (higher V _max) for O₂ reduction than the two other oxidases. It is postulated that the pyruvate oxidase acts as a scavenger for O₂, leading to the non-toxic reduction product H₂O, and thus functions as a defense mechanism against oxygen toxicity when the organism is exposed to aerobic condition.Abbreviations PY peptone-yeast extract - PYG PY-glucose - PN pyridine nucleotide - PNH reduced PN - CCCP carbonylcyanide m-chlorophenylhydrazone - DNP 2.4-dinitrophenol     

Epr Spectra of Dmpo Spin Adducts of Superoxide and hydroxyl Radicals in Pyridine

Electron spin resonance spectroscopy and the spin trapping technique were used to study the formation of the superoxide radical in pyridine. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was employed as a trapping agent. Superoxide radical was generated using chemical (potassium superoxide) and photochemical methods with anthralin, benzanthrone, rose bengal, 1,8-dihydroxyanthraquinone and zinc tetraphenylporphyrine as photoactive pigments. Hyperfine coupling (hf) constants for DMPO/O₂- were determined to be a_N = 12.36 G, a^β_H= 9.85G, a^γ_H = 1.34 G. The a_N and a^β_H constants are in good agreement with values calculated from a previously determined relationship between hf constants and solvent acceptor number (Reszka et al., (1992) Free Radical Res. Commun., in press). When concentrated hydrogen peroxide was added to DMPO in pyridine a similar EPR spectrum was observed. It is suggested that in this case the DMPO/'O₂H adduct is formed by nucleophilic addition of H₂O₂ to DMPO to give a hydroxylamine, followed by oxidation to the respective nitroxide. The EPR spectrum observed when tetrapropylammonium hydroxide and H₂O₂ were added to DMPO in pyridine had hf couplings a_N = 13.53 G, a^β_H = 11.38 G, a^γ_H = 0.79 G and it was assigned to a DMPO/'OH adduct. This assignment was based on similarity of this spectrum to the one produced by UV photolysis of hydrogen peroxide and DMPO in aqueous solution and subsequent transfer to pyridine.     

Synthesis and Biological Activity of 5-Azacytosine Nucleosides Derived from 4-Thio-2-Deoxy-L-threo-Pentofuranose and 4-Thio-2-Deoxy-D-erythro-Pentofuranose

Abstract

1-O-Acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-_d-erythro-pentofuranose and 2-deoxy-1,3,5-tri-O-acetyl-4-thio-_l-threo-pentofuranose were coupled with 5-azacytosine to obtain α and β anomers of nucleosides.     

Crystal structures of two cyclic pseudopentapeptides containing ψ[CH2S] and ψ[CH2SO] backbone surrogates

The solid state conformations of cyclo[Gly–Proψ[CH₂S]Gly–D –Phe–Pro] and cyclo[Gly–Proψ[CH₂–(S)–SO]Gly–D –Phe–Pro] have been characterized by X-ray diffraction analysis. Crystals of the sulfide trihydrate are orthorhombic, P2₁2₁2₁, with a = 10.156(3) Å, b = 11.704(3) Å, c = 21.913(4) Å, and Z = 4. Crystals of the sulfoxide are monoclinic, P2₁, with a = 10.662(1) Å, b = 8.552(3) Å, c = 12.947(2) Å, β = 94.28(2), and Z = 2. Unlike their all-amide parent, which adopts an all-trans backbone conformation and a type II β-turn encompassing Gly-Pro-Gly-D -Phe, both of these peptides contain a cis Gly¹-Pro² bond and form a novel turn structure, i.e., a type II′ β-turn consisting of Gly–D –Phe–Pro–Gly. The turn structure in each of these peptides is stabilized by an intramolecular H bond between the carbonyl oxygen of Gly¹ and the amide proton of D -Phe⁴. In the cyclic sulfoxide, the sulfinyl group is not involved in H bonding despite its strong potential as a hydrogen-bond acceptor. The crystal structure made it possible to establish the absolute configuration of the sulfinyl group in this peptide. The two crystal structures also helped identify a type II′ β-turn in the DMSO-d₆ solution conformers of these peptides. © 1993 John Wiley & Sons, Inc.     

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

Purification,Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P₅₀) and 1.96 (n value), and a small Bohr effect (δH⁺ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O₂ affinity at P₅₀ of 2.5 mmHg which may assure efficient utilization of the lung O₂ reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl₂·6H₂O] solution at a pH of 7.0–8.5.     

A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species

Various aspects of the biochemistry of photosynthetic carbon assimilation in C₃ plants are integrated into a form compatible with studies of gas exchange in leaves. These aspects include the kinetic properties of ribulose bisphosphate carboxylase-oxygenase; the requirements of the photosynthetic carbon reduction and photorespiratory carbon oxidation cycles for reduced pyridine nucleotides; the dependence of electron transport on photon flux and the presence of a temperature dependent upper limit to electron transport. The measurements of gas exchange with which the model outputs may be compared include those of the temperature and partial pressure of CO₂(p(CO₂)) dependencies of quantum yield, the variation of compensation point with temperature and partial pressure of O₂(p(O₂)), the dependence of net CO₂ assimilation rate on p(CO₂) and irradiance, and the influence of p(CO₂) and irradiance on the temperature dependence of assimilation rate.Abbreviations RuP₂ ribulose bisphosphate - PGA 3-phosphoglycerate - C=p(CO₂) partial pressure of CO₂ - O=p(O₂) partial pressure of O₂ - PCR photosynthetic carbon reduction - PCO photorespiratory carbon oxidation     

Studies on the conformations and interactions of elastin. Proton magnetic resonance of the repeating tetramer

Proton magnetic resonance studies at 220 MHz were carried out on synthetic polymers of the repeating tetrapeptide of elastin. Temperature dependence and solvent-mixture dependence of peptide proton chemical shifts were determined for both linear polymers, N-formyl-(Val-Pro-Gly₁-Gly₂)_n-Val OMe where n ? 8 and 40, and for cyclic polymers (Val-Pro-Gly₁-Gly₂)_n, where n = 3 and 4. The Gly₂ NH was found to be solvent shielded. In addition, by studying the polymers Boc-Val-Pro-Gly₁-Gly₂-OH, H-Pro-Gly₁-Gly₂-Val OMe, H(Pro-Gly₁-Gly₂-Val)₃OH, and others, it was demonstrated that the Val C–O immediately preceding the Gly₂ NH in the sequence was required for solvent shielding. Also the Gly₂ NH resonance is found at higher field than the Gly₁ NH resonance. This provides the basis for proposing a β turn in which the Pro and Gly₁ residues form the corners, i.e., residues i + 1 and i + 2, and in which the Gly₂ NH, residue i + 3 hydrogen bonds to the carbonyl of residue i, the Val residue. Studies on methanol–water solvent systems indicated retention of the β turn as a significant conformational feature. This suggests that the β turn occurs in the elastic fiber, which contains about 60% water but which utilizes association of hydrophobic groups as a primary force in fibrogenesis.     

Generation of reducing prower in chemosynthesis

Summary Cell-free preparations from T. neapolitanus catalyzed an ATP-dependent reduction of pyridine nucleotides by thiosulfate. The reduction of flavins by thiosulfate was also observed to be an energy-linked process. Optimal reaction occurred at pH 7.3–7.5 in the presence of 7 mM S₂O₃ ⁼, 1.5 mM ATP and 0.7 mM NAD⁺ or NADP⁺. The enzyme(s) catalyzing the energy-linked reactions appear to reside in the 144000 x g supernatant fraction since washed particles failed to catalyze the ATP driven NAD⁺ reduction by S₂O₃ ⁺; the cell-free preparations contained, however, S₂O₃ ⁼ oxidase and ferro-cytochrome c: O₂ oxidoreductase activities. The ATP-driven reduction of flavins or that of the pyridine nucleotides was inhibited bythe inhibitors that intersect the electron transport chain in the flavin or that of the cytochrome b and c regions. In the flavin-inhibited system, quinones could substitute as electron bypass carriers for the reduction of pyridine nucleotides. Uncouplers of oxidative phosphorylation and oligomycin inhibited the energy-transfer reactions. A utilization of 2 to 3 ATP equivalents was observed for the reduction of each equivalent of NAD⁺. Such observations indicate that the T. neapolitanus system operated with an efficiency of approximately 80% with respect to the utilization of energy for the generation of reducing power.Non-standard abbreviations HQNO 2-n-hyptyl-4-hydroxyquinoline N-oxide - TTFA Thenoyl triflouroacetone - CCCP m-chlorocarbonylcyanide-phenylhydrazone - DNP 2,4-dinitrophenol     

Conformational change of poly-N epsilon-glutaryl-L-lysine and poly-N epsilon-succinyl-L-lysine in aqueous salt solutions

The conformational changes of poly-N^ε-glutaryl-L -lysine (PGL) and poly-N^ε-succinyl-L -lysine (PSL) in various salt solutions were studied by use of ORD and potentiometric titration measurements. The addition of alkali metal salts to the fully ionized PGL or PSL solution caused helix formation. The helical content of the polymers increases in the following sequences: at salt concentration 0–2 M, CsCl < KCl < LiCl < NaCl; and at 2–3 M, LiCl < CsCl < KCl ～ NaCl. The preferential binding of the solvent components with various alkali metal salts of PGL or PSL was measured in LiCl, NaCl, and KCl solutions by means of equilibrium dialysis and differential refractometry. It was found that with increasing salt concentration, the polymers were preferentially hydrated in NaCl and KCl soultions; however the salt was preferentially bound to the polymers in LiCl solution. Such preferential binding was suggested to be closely related to conformational change. The addition of CaCl₂ to polymer solutions led to the stabilization of the helical structure of PGL or PSL.     

Aspects of carbon metabolism in Chloroflexus

When fluoroacetate was added to aerobic, washed cells of Chloroflexus, O₂ uptake was strongly inhibited and citrate accumulated. Under anaerobic conditions in the light, fluoroacetate inhibited CO₂ uptake and caused citrate accumulation. The results are taken as evidence for the operation of a tricarboxylic acid cycle in Chloroflexus both under aerobic conditions in the dark and anaerobically in the light. 2. Organic compounds are assimilated into the storage materials polyglucose and poly--hydroxybutyric acid by washed cells of Chloroflexus. The type of storage product formed from acetate depends upon the availability of reducing power. 3. Low activities of the key enzymes of the reductive pentose phosphate cycle, ribulose-1,5-bisphosphate carboxylase and phosphoribulokinase were detected in cell free extracts of photoheterotrophically grown Chloroflexus.Abbreviations RuBP Ribulose-1,5-bisphosphate - TCA tricarboxylic acid - PHB poly--hydroxybutyric acid     

Studies on the metal—amide bond. XVII. The crystal and molecular structure of the α-form of aqua[N,N′-bis(2′-pyridinecarboxamido)-1,3-propane] copper(II) dihydrate

α-Aqua[N,N′-bis(2′-pyridinecarboxamido)-1,3-propane]copper(II) dihydrate, C₁₅H₂₀N₄O₅Cu, is monoclinic, space group P2₁/c, with a = 11.719(2), b = 13.092(2), c = 12.663(2) Å, β = 119.56(1)°, Z = 4. The structure was refined to R = 0.026 for 2398 diffractometer data using full-matrix least-squares methods. The copper atom is five-coordinate with the N₄-tetradentate ligand encompassing the base of a distorted square-based pyramid which is appreciably distorted towards a trigonal bipyramid [average Cu-N(amide) 1.950(2), Cu-N(pyridine) 2.043(2) Å, N(amide)-Cu-N(amide) 94.5(1), N(pyridine)-Cu-N(pyridine) 100.2(1)°] and with the copper atom lying 0.27 Å above the N₄ plane towards the apical water molecule [Cu-O 2.236(2) Å]. The central six-membered chelate ring adopts a skewed boat conformation and the enforced strain in the molecule results in non-planar distortions in the pyridine rings with only small distortions in the amide groups. The molecules pack in sheets parallel to (10$\text{1}$) and the hydrogen-bonding network involves the water molecules and the amide oxygen atoms of the ligand.     

Conformation of oligopeptides with L-leucyl-L-leucyl-L-alanyl and L-methionyl-L-methionyl-L-leucyl repeating units

The conformation of oligopeptides with hydrophobic side chains, Nps-(L -Leu-L -Leu-L -Ala)_n-OEt and Nps-(L -Met-L -Met-L -Leu)_n-OEt(n = 1–6), in the solid state, obtained either by evaporation of the solvent or by precipitation with diethyl ether from a 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) solution, has been studied with ir spectroscopy and x-ray powder-diffraction measurements. The conformation of these peptides in the HFIP solution has been studied by CD spectroscopy. Due to a strong preference of the amino acids to form an α helix, the peptides begin forming α helices at the dodecapeptide in the HFIP solution, and in the solid state by evaporation. In the solid state, with precipitation, the α-helical conformation is first observed at the octadecapeptide and the lower peptides assume a β structure. The conformational change, from the α helix to the β structure of the peptides with 12 to 15 amino acid residues, during the precipitation process, is due to a strong tendency of the amino acids to form the β-structure in rather short peptide lengths.     

The beta conformation of polypeptides of valine, isoleucine, and threonine in solution and solid-state: optical and infrared studies.

Water-soluble polypeptides of L -valyl and L -isoleucyl residues flanked with DL -lysyl blocks [poly(DL Lys · HCl)_x–poly(L Val)_y–poly(DL Lys · HCl)_x, poly(DL Lys · HCl)_x–poly-(L Ile)_y–poly(DL Lys · HCl)_x] and homopoly(L -threonine) were prepared. The β conformation of these polymers in water, as well as in aqueous methanol, was confirmed by infrared spectroscopy and circular dichroism studies. The optical properties of these valyl and isoleucyl polypeptides were quite different from those of previously reported synthetic homopolypeptides in the β structure. Their differences could be explained by the presence of a “single extended β chain” without either intra- or interchain association.     

Nitrogen-limited continuous culture ofRhodopseudomonas capsulata growing photosynthetically or heterotrophically under low oxygen tensions

A continuous culture apparatus is described, which allows cultivation of photosynthetic bacteria anaerobically in the light and semiaerobically in the dark at constant oxygen tensions. The growth-parameters 1. substrate concentration at half maximum growth rate (K _s) and 2. yield (Y) forRhodopseudomonas capsulata are calculated.An automatic control system of the oxygen partial pressure in the culture medium is elaborated. It is shown, that the discontinuous regulation with a control unit in connection with two magnetic valves, which give short pulses of either oxygen-free gas or gas mixed with oxygen, is an economic, practicable and reliable method.The yield coefficientY during growth limited by ammonium sulfate is variable due to the synthesis of nitrogen independent metabolites, such as poly--hydroxybutyrate. The storage of poly--hydroxybutyrate in continuous culture is a function of both the actual concentration of ammonia and of theC/N ratio. At very low growth rates (1/6 µ _m ) the poly--hydroxybutyrate content increases amounting to 33% of the dry weight.In semiaerobically dark grown cells (pO₂: 5 mm Hg) the protein and bacteriochlorophyll content increased definitely on dry weight basis with increasing growth rates. In contrast, in anaerobic light cultures only a small increase of the bacteriochlorophyll level but no change of the protein content per dry weight of cells was observed at increasing growth rates.