Mechanism of nitrosylmyoglobin autoxidation: temperature and oxygen pressure effects on the two consecutive reactions

As shown by singular value decomposition and global analysis of the absorption spectra, oxidation of nitrosylmyoglobin, MbFe(II)NO, by oxygen occurs in two consecutive (pseudo) first-order reactions in aqueous air- saturated solutions at physiological conditions (pH 7.0, I=0.16 m (NaCl)). Both reaction steps have a large temperature dependence with the following activation parameters: DeltaS++(1) = 121+/-7 and DeltaS++(1) = 23+/-29; and DeltaS++(2) = 88+/-14 kJ mol(-1) and DeltaS++(2)-63+/-51 J(-1) K(-1) mol(-1) at 25 degrees C for the first and second step, respectively. At physiological temperature, the initial reaction is faster, while at lower temperatures, the first reaction is slower and rate-determining. The rate of the first reaction is linearly dependent on oxygen pressure at lower pressures, while for oxygen pressures above atmospheric, the rate exhibits saturation behaviour. The second reaction is independent of oxygen pressure. The rate of the second reaction increases when oxymyoglobin is added. In contrast, the rate of the first reaction is independent of the presence of oxymyoglobin. The observed kinetics are in agreement with a reaction mechanism in which the nitric oxide that is initially bound to the Fe(II) centre of myoglobin is displaced by oxygen in a reversible ligand-exchange reaction prior to an irreversible electron transfer. The ligand-exchange process is dissociative in nature and depends bond breaking, and nitric oxide is suggested to be trapped in a protein cavity. The absorption spectrum of the intermediate, as resolved from the global analysis, is in agreement with a peroxynitrite complex, and the initial process must involve partial electron transfer.     

Radiolysis of aqueous solutions of 1,1- and 1,2-dichloroethane

The yields of chloride ion and molecular hydrogen were determined in the gamma, the fast electron, and the 5 MeV helium ion radiolysis of deaerated and aerated aqueous solutions of 1,1- and 1,2-dichloroethane. In deaerated solutions irradiated with gamma-rays or fast electrons, the yield of chloride ion increases while the yield of molecular hydrogen decreases with increasing dichloroethane concentration. These results are due to the quantitative reaction of both the hydrated electron and the hydrogen atom with the dichloroethane to produce chloride ions. The yield of chloride ions is significantly larger in aerobic than in anaerobic conditions and is dependent upon the dose rate. Formation of peroxyl radicals by the reaction of molecular oxygen with chlorinated hydrocarbon radicals and their subsequent chemistry are responsible for the observed increase in chloride ions. The yield of chloride ion with 5 MeV helium ions is smaller than with gamma irradiation, while the yield of molecular hydrogen is larger reflecting the higher density of reactive species and consequent increase in intratrack reactions in a helium ion track compared to a gamma-ray track.     

Molecular dynamics study on the solvent dependent heme cooling following ligand photolysis in carbonmonoxy myoglobin

The time scale and mechanism of vibrational energy relaxation of the heme moiety in myoglobin was studied using molecular dynamics simulation. Five different solvent models, including normal water, heavy water, normal glycerol, deuterated glycerol and a nonpolar solvent, and two forms of the heme, one native and one lacking acidic side chains, were studied. Structural alteration of the protein was observed in native myoglobin glycerol solution and native myoglobin water solution. The single-exponential decay of the excess kinetic energy of the heme following ligand photolysis was observed in all systems studied. The relaxation rate depends on the solvent used. However, this dependence cannot be explained using bulk transport properties of the solvent including macroscopic thermal diffusion. The rate and mechanism of heme cooling depends upon the detailed microscopic interaction between the heme and solvent. Three intermolecular energy transfer mechanisms were considered: (i) energy transfer mediated by hydrogen bonds, (ii) direct vibration-vibration energy transfer via resonant interaction, and (iii) energy transfer via vibration-translation or vibration-rotation interaction, or in other words, thermal collision. The hydrogen bond interaction and vibration-vibration interaction between the heme and solvent molecules dominates the energy transfer in native myoglobin aqueous solution and native myoglobin glycerol solutions. For modified myoglobin, the vibration-vibration interaction is also effective in glycerol solution, different from aqueous solution. Thermal collisions form the dominant energy transfer pathway for modified myoglobin in water solution, and for both native myoglobin and modified myoglobin in a nonpolar environment. For native myoglobin in a nonpolar solvent solution, hydrogen bonds between heme isopropionate side chains and nearby protein residues, absent in the modified myoglobin nonpolar solvent solution, are key interactions influencing the relaxation pathways.     

Photo-induced reduction of flavin mononucleotide in aqueous solutions

The photo-induced reduction of flavin mononucleotide (FMN) in aqueous solutions is studied by absorption spectra measurement under aerobic and anaerobic conditions. Samples without exogenous reducing agent and with the exogenous reducing agents ethylene-diamine-tetraacetic acid (EDTA) and dithiothreitol (DTT) are investigated. Under anaerobic conditions the photo-induced reduction with and without reducing agents is irreversible. Under aerobic conditions the photo-reduction without added reducing agent is small compared to the photo-degradation, and the photo-reduction of FMN by the reducing agents is reversible (re-oxidation in the dark). During photo-excitation of FMN the dissolved oxygen is consumed by singlet oxygen formation and subsequent chemical reaction. After light switch-off slow re-oxidation (slow absorption recovery) occurs due to air in-diffusion from surface. EDTA degradation by FMN excitation leads to oxygen scavenging. The quantum efficiencies of photo-reduction under aerobic and anaerobic conditions are determined. The re-oxidation of reduced FMN under aerobic conditions and due to air injection is investigated.     

Effects of supercharging reagents on noncovalent complex structure in electrospray ionization from aqueous solutions

The effects of two supercharging reagents, m-nitrobenzyl alcohol (m-NBA) and sulfolane, on the charge-state distributions and conformations of myoglobin ions formed by electrospray ionization were investigated. Addition of 0.4% m-NBA to aqueous ammonium acetate solutions of myoglobin results in an increase in the maximum charge state from 9+ to 19+, and an increase in the average charge state from 7.9+ to 11.7+, compared with solutions without m-NBA. The extent of supercharging with sulfolane on a per mole basis is lower than that with m-NBA, but comparable charging was obtained at higher concentration. Arrival time distributions obtained from traveling wave ion mobility spectrometry show that the higher charge state ions that are formed with these supercharging reagents are significantly more unfolded than lower charge state ions. Results from circular dichroism spectroscopy show that sulfolane can act as chemical denaturant, destabilizing myoglobin by ∼1.5 kcal/mol/M at 25 °C. Because these supercharging reagents have low vapor pressures, aqueous droplets are preferentially enriched in these reagents as evaporation occurs. Less evaporative cooling will occur after the droplets are substantially enriched in the low volatility supercharging reagent, and the droplet temperature should be higher compared with when these reagents are not present. Protein unfolding induced by chemical and/or thermal denaturation in the electrospray droplet appears to be the primary origin of the enhanced charging observed for noncovalent protein complexes formed from aqueous solutions that contain these supercharging reagents, although other factors almost certainly influence the extent of charging as well.     

OXYGEN QUENCHING IN MICELLAR SOLUTION AND ITS EFFECT ON THE PHOTOCYCLIZATION OF N-METHYLDIPHENYLAMINE

Abstract— Oxygen effects on the photocyclization of N-methyldiphenylamine to N-methylcarbazole were investigated in n-hexane, water, and aqueous surfactant solutions by steady state irradiations and flash photolysis measurements. The reaction sequence in micelles was found to involve the same intermediate steps as in homogeneous solutions. In aerated micellar solutions, the quantum yield of N-methylcarbazole is significantly higher than in n-hexane, while the rate constants of the unimolecular reaction steps show no solvent dependence. The bimolecular dehydrogenation of the intermediate 4a, 4b-dihydro-N-methylcarbazole by oxygen is enhanced in aqueous and micellar solutions, whereas the quenching rate of triplet intermediates by oxygen was not affected. The lesuhs are interpreted using a dispersed phase model of micellar solutions. Special 'micellar effects' need not be invoked since the dependence of the quantum yield on the solvent is shown to be due to the difference in the overall oxygen concentration.     

Evidence for the role of myoglobin in facilitating oxygen transport

Cyclic voltammetric studies of the heterogeneous electron-transfer reactions of myoglobin under aerobic and anaerobic conditions are reported. Evidence for a role of myoglobin that has not been previously measured directly, namely, facilitation of oxygen transport, is presented. It is suggested that one molecule of oxygen can be contained within the structure of the oxidized form of myoglobin, but is not co-ordinated to the heme iron. Reduced myoglobin binds one molecule of oxygen to the heme iron but no reports have been found that suggest that the oxidized form of myoglobin binds to, or contains a molecule of, oxygen.     

One-electron reductive template-directed ligation of oligodeoxynucleotides possessing a disulfide bond

Hypoxic X-radiolysis of diluted aqueous solutions was performed to generate hydrated electrons that induced one-electron reduction of oligodeoxynucleotides (ODNs) possessing a disulfide bond. Upon hypoxic irradiation of dinucleotides, two forms of dinucleotides were produced via intermolecular exchange of the disulfides and ligation that proceeded with a multiple turnover. In contrast to the efficient reaction induced by hypoxic irradiation, the reaction efficiency was dramatically decreased when irradiation was performed under aerobic conditions, presumably due to capturing reactive hydrated electrons by molecular oxygen. We subsequently applied these unique reaction characteristics to template-directed ligation. In the presence of a complementary template ODN, two ODNs possessing a disulfide bond produced a prescribed ODN with high regioselectivity via interstrand crossing upon hypoxic irradiation.     

PHOTOCHEMICAL REACTIONS IN RIGID MEDIA-I. THE EFFECT OF GELATIN ON THE AEROBIC PHOTOCHEMISTRY OF RIBOFLAVIN IN AQUEOUS SOLUTION

Abstract— The addition of potassium iodide to dilute aqueous solutions of riboflavin reduced both the rate of the aerobic photolysis and the fluorescence quantum yield of riboflavin in the same proportions. This indicated that under these conditions the photolysis proceeded from the singlet excited state. The addition of gelatin to aqueous solutions of riboflavin also reduced the rate of the aerobic photolysis but increased slightly the quantum yield of fluorescence. The rates and the fluorescence of solutions of riboflavin to which gelatin had been added were also reduced by the addition of potassium iodide but in this case the effect on the rate was proportionately greater than the effect on the fluorescence. The data suggests that in the presence of gelatin the mechanism of the reaction is changed and that the triplet state becomes more important.     

Bioelectrochemical dehalogenations via direct electrochemistry of poly(ethylene oxide)-modified myoglobin

Catalytic reductive dehalogenation of hexachloroethane (HCE) was shown to be possible using a graphite electrode coated with a film of the poly(ester sulfonic acid) containing myoglobin (Mb). The effectiveness of the dehalogenation was limited by the solubility of HCE in aqueous solutions. In order to produce an electrode that could dehalogenate HCE in non-aqueous solutions, Mb was chemically modified by addition of poly(ethylene oxide) (PEO). The PEO–Mb–AQ29D modified electrode was found to be suitable for the reductive dehalogenation of HCE in ethanolic solutions. The catalytic response was shown to be linearly dependent on the bulk concentration of HCE.     

Water-stable biphasic nanocolloids with potential use as anisotropic imaging probes

Electrified co-jetting of two aqueous polymer solutions followed by a thermal cross-linking step was used to create water-stable biphasic nanocolloids. For this purpose, aqueous solution mixtures of poly(acrylamide-co-acrylic acid) and poly(acrylic acid) were employed as jetting solutions. When the biphasic nanocolloids created by side-by-side electrified co-jetting were thermally treated, a cross-linking reaction occurred between amide groups and carboxylic groups to form stable imide groups. Infrared spectroscopy was employed to monitor the reaction. The quality and the integrity of the resulting biphasic nanocolloids were confirmed by confocal laser scanning microscopy, flow cytometry analysis, and dynamic light scattering. Selective encapsulation of two biomolecules in each phase of the biphasic colloids was maintained even after thermal reaction and suspension in aqueous environment. Well-dispersed spherical colloids with stable dye loadings in each hemisphere were kept intact without aggregation or dissolution for several weeks. Finally, biphasic nanocolloids were selectively surface-modified with a biotin-dextran resulting in water-stable particles to ensure binding of proteins only to a single hemisphere.     

PHOTOCHEMICAL and ENZYMATIC REDOX TRANSFORMATIONS OF REDUCED FORMS OF COENZYME NADP+

The two reduced forms of NADP+, NADPH and its dimer (NADP)2, on irradiation in aqueous medium at 365 nm, are converted to enzymatically active NADP+, with accompanying formation of H2O2. The rate photooxidation of NADPH is strongly dependent on the presence of oxygen, but that of (NADP)2 is similar under aerobic and anaerobic conditions. In the presence of oxygen, but not in its absence, O2-. is an intermediate in the reaction. Generation of H2O2 under anaerobic conditions, confirmed by the fact that presence of peroxidase in irradiated solutions of (NADP)2 enhances photooxidation of the latter, is ascribed to attack on water of the excited dimer. Under anaerobic conditions at pH 9.5, Fe(EDTA)2+ and Fe(CN)4-(6) increase the rate of photooxidation of NADP dimer two-fold. gamma-Irradiation of (NADP)2 at pH 9.5 in the presence of N2O results in 80% conversion to enzymatically active NADP+. A mechanism for photooxidation of (NADP)2 under anaerobic conditions is suggested, and some relevant biological implications are presented.     

Copper(II)-catalyzed oxidation of d-alpha-tocopherol by oxygen in aqueous solution

alpha-Tocopherol (alpha-Toc) was solubilized in aqueous solutions using 13 solubilizing agents and the products of oxidation by oxygen in the presence and the absence of Cu(II) were analyzed by HPLC. In the presence of Cu(II), the oxidation was accelerated and 5-formyl-7,8-dimethyltocol and alpha-tocoquinone were the major oxidation products. Their yields greatly increased in the presence of Cu(II). The yields and the rates of formation of the products were dependent on the properties of solubilizing agents and other conditions as well as the presence of Cu(II) or other metal ions. It is suggested that slight changes in the structure of the solubilizing agents affect the course of the reaction.     

NaOH溶液中碱浓度、氧气压力以及温度对Pt电极上氧气还原反应的影响

设计制作一种新型耐压电化学池并采用循环伏安(CV)和线性扫描伏安(LSV)技术系统研究了碱浓度、氧气压力以及温度对NaOH溶液中氧气还原反应(ORR)的影响. 研究结果表明,碱浓度、氧气压力和温度对ORR动力学和热力学都有很大的影响. 随着碱浓度增大,ORR过程逐渐由2 电子(生成HO₂^-)转为1 电子(生成O₂^-)反应,并且由于氧气溶解度减小和体系粘度增大ORR过程受到很大抑制. 增大压力可以明显增加氧气溶解度,从而从动力学上促进ORR过程;同时计算得到了氧气在不同浓度NaOH溶液中的亨利系数. 随着介质温度升高,由于氧气反应活性增强、扩散系数增大和溶解度减小的共同作用,表现出在给定浓度下存在一最佳温度使得ORR峰电流达到最大.     

An oxidative cross-dehydrogenative-coupling reaction in water using molecular oxygen as the oxidant: vanadium catalyzed indolation of tetrahydroisoquinolines

An aerobic oxidative cross-dehydrogenative coupling reaction between sp(3) C-H and sp(2) C-H bonds is developed by employing a vanadium catalyst (10 mol%) in an aqueous medium using molecular oxygen as the oxidant. This environmentally benign strategy exhibits larger substrate scope and shows high regioselectivity.     

Radiolysis of aqueous solutions of thiamine

The results of the radiolysis of aqueous solutions of thiamine (vitamin B₁) are presented. The yields for decomposition of thiamine and the product of radiolytic products were determined. The G values decrease as the dose increases. Some radiolytic products were identified. Decomposition of thiamine was slightly dependent on the presence of oxygen and on the pH of the solution. At pH 4.4 with a concentration of 2.5 × 10⁻⁴ mol L⁻¹ of thiamine in an oxygen free aqueous solution, the G⁰ value for decomposition is 5.0.     

Release of nitric oxide and iodine to the atmosphere from the freezing of sea-salt aerosol components

The known room-temperature, solution-phase reaction between nitrite ions and iodide ions, which occurs in acidic conditions (pH < 5.5), is shown to be accelerated when neutral aqueous solutions are frozen. The reaction is proposed to occur in liquid "micropockets" within the ice structure at temperatures between the freezing point and the eutectic temperature. The products, nitric oxide and molecular iodine, are known to play significant roles in atmospheric compositional change, and therefore, the results obtained here, which are not dependent on acidification, may impact on observed snowpack chemistry. Investigation of the effect of oxygen on the chemical processing indicates that a chain reaction mechanism is operative.     

Protein Conformation and Supercharging with DMSO from Aqueous Solution

The efficacy of dimethyl sulfoxide (DMSO) as a supercharging reagent for protein ions formed by electrospray ionization from aqueous solution and the mechanism for supercharging were investigated. Addition of small amounts of DMSO to aqueous solutions containing hen egg white lysozyme or equine myoglobin results in a lowering of charge, whereas a significant increase in charge occurs at higher concentrations. Results from both near-UV circular dichroism spectroscopy and solution-phase hydrogen/deuterium exchange mass spectrometry indicate that DMSO causes a compaction of the native structure of these proteins at low concentration, but significant unfolding occurs at ~63% and ~43% DMSO for lysozyme and myoglobin, respectively. The DMSO concentrations required to denature these two proteins in bulk solution are ~3–5 times higher than the concentrations required for the onset of supercharging, consistent with a significantly increased concentration of this high boiling point supercharging reagent in the ESI droplet as preferential evaporation of water occurs. DMSO is slightly more basic than m-nitrobenzyl alcohol and sulfolane, two other supercharging reagents, based on calculated proton affinity and gas-phase basicity values both at the B3LYP and MP2 levels of theory, and all three of these supercharging reagents are significantly more basic than water. These results provide additional evidence that the origin of supercharging from aqueous solution is the result of chemical and/or thermal denaturation that occurs in the ESI droplet as the concentration of these supercharging reagents increases, and that proton transfer reactivity does not play a significant role in the charge enhancement observed.     

Design of an Oxygen-Tolerant Photo-RAFT System for Protein-Polymer Conjugation Achieving High Bioactivity

Protein-polymer conjugates have significant potential in pharmaceutical and biomedical applications. To enable their widespread use, robust conjugation techniques are crucial. This study introduces a photo-initiated reversible addition-fragmentation chain-transfer (Photo-RAFT) polymerization system that exhibits excellent oxygen tolerance. This system allows for the synthesis of protein-polymer conjugates with high bioactivity under mild and aerobic conditions. Three photocatalytic systems utilizing Eosin Y (EY) as the photocatalyst with two different cocatalysts (ascorbic acid and triethanolamine) were investigated, each generating distinct reactive oxygen species (ROS) such as singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals. The impact of these ROS on three model proteins (lysozyme, albumin, and myoglobin) was evaluated, demonstrating varying bioactivities based on the ROS produced. The EY/TEOA system was identified as the optimal photo-RAFT initiating system, enabling the preparation of protein-polymer conjugates under aerobic conditions while maintaining high protein enzymatic activity. To showcase the potential of this approach, lysozyme-poly(dimethylaminoethyl acrylate) conjugates were successfully prepared and exhibited enhanced antimicrobial property against Gram-positive and Gram-negative bacteria.     

Heavy Atom Effects in Tellurapyrylium Dyes Useful in Photodynamic Therapy and Catalytic Generation of H2O2

Abstract

The large rate of intersystem crossing between singlet and triplet states of tellurapyrylium dyes leads to efficient generation of singlet oxygen in irradiated airsaturated aqueous solutions containing these dyes. One reaction of tellurapyrylium dyes with singlet oxygen and water is the formation of dihydroxy tellurane [tellurium(IV)] species. We have found that the photochemical generation of dihydroxy telluranes is reversible thermally. The tellurapyrylium dye is regenerated while a molecule of hydrogen peroxide is produced. The thermal generation of hydrogen peroxide coupled with a photochemical generation of singlet oxygen allows a catalytic cycle to be devised for the conversion of oxygen and water to hydrogen peroxide. The dihydroxy telluranes are efficient two-electron oxidizing agents and can be used as catalysts to accelerate reactions using hydrogen peroxide as a two-electron oxidizing agent. Examples of tellurapyrylium dye-mediated reactions of hydrogen peroxide include reactions of leucodyes normally oxidized by horseradish peroxidase and hydrogen peroxide. These processes lead to thermal and photochemical reactions that are potentially cytotoxic following the generation of singlet oxygen in photodynamic therapy. The regeneration of the original catalyst allows repeated treatment from a single dose.