Thermal inactivation, denaturation and aggregation of mitochondrial aspartate aminotransferase

A comparative study of thermal denaturation and inactivation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been carried out (10 mM Na phosphate buffer, pH 7.5). Analysis of the data on differential scanning calorimetry shows that thermal denaturation of mAAT follows the kinetics of irreversible reaction of the first order. The kinetics of thermal inactivation of mAAT follows the exponential law. It has been shown that the inactivation rate constant (k_in) is higher than the denaturation rate constant (k_den). The k_in/k_den ratio decreases from 28.8 ± 0.1 to 1.30 ± 0.09 as the temperature increases from 57.5 to 77 °C. The kinetic model explaining the discrepancy between the inactivation and denaturation rates has been proposed. The size of the protein aggregates formed at heating of mAAT at a constant rate (1 °C min^{− 1}) has been characterized by dynamic light scattering.     

Kinetics of Reduction of Hypervalent Iron in Myoglobin by Crocin in Aqueous Solution

Crocin in aqueous solution is oxidized by ferrylmyoglobin, MbFe(IV)=O, in a second order reaction with k = 183 1 · mol^-1 · s^-1, AH^‡₂₉₈ = 55.0 kJ · mol^-1, and ΔLS^‡₂₉₈ = -17 J · mol^-1 K^-1 (pH = 6.8, ionic strength 0.16 (NaCl), 25°C), as studied by stopped-flow spectroscopy. The reaction has 1:1 stoichiometry to yield metmyoglobin, MbFe(III), and has AG^o = -11 kJ · mol^-1, as calculated from the literature value E⁰ = +0.85 V (pH = 7.4) vs. NHE for MbFe(IV)=O/MbFe(III) and from the half-peak potential +0.74 V (vs. NHE in aqueous 0.16 NaCl, pH = 7.4) determined by cyclic voltammetry for the one-electron oxidation product of crocin, for which a cation radical structure is proposed and which has a half-peak potential of +0.89 V for its formation from the two-electron oxidation product of crocin. The fer-rylmyoglobin protein-radical, MbFe(IV)=O, reacts with crocin with 2:l stoichiometq to yield MbFe(IV)= 0, as determined by ESR spectroscopy, in a reaction faster than the second order protein-radical generating reaction between H₂O₂ and MbFe(III), for which latter reaction k = 137 L · mol^-1 · s^-1, ΔH^‡₂₉₈ = 51.5 kJ · mol^-1, and ΔH^‡₂₉₈ = -31 J · mol^-1 · K^-1 (pH = 6.8, ionic strength = 0.16 (NaCI), 25°C) was determined. Based on the difference between the stoichiometry for the reaction between crocin and each of the two hypervalent forms of myoglobin, it is concluded in agreement with the determined half peak reduction potentials, that the crocin cation radical is less reducing compared to crocin, as the cation radical can reduce the protein radical but not the iron(IV) centre in hypervalent myoglobin.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Enantioselective cleavage of activated amino acid esters promoted by chiral palladacycles

The ester cleavage of R- and S-isomers N-CBZ-leucine p-nitrophenyl ester intermolecularly catalyzed by R- (a) and S-stereoisomers (b) of the Pd(II) metallacycle [Pd(C₆H₄C^*HMeNMe₂)Cl(py)] (3) follows the rate expression k_obs = k_o + k_cat [3], where the rate constants k_cat equal 25.8 ± 0.4 and 7.6 ± 0.5 dm³ mol⁻¹ s⁻¹ for the S- and R-ester, respectively, in the case of 3a, but are 5.7 ± 0.6 and 26.7 ± 0.5 dm³ mol⁻¹ s⁻¹ for the S- and R-ester, respectively, in the case of 3b (pH 6.23 and 25°C). Thus, the best catalysis occurs when the asymmetric carbons of the leucine ester and Pd(II) complex are R and S, or S and R configured, respectively. Molecular modeling suggests that the stereoselection results from the spatial interaction between the CH₂CHMe₂ radical of the ester and the -methyl group of 3. A hydrophobic/stacking contact between the leaving 4-nitrophenolate and the coordinated pyridine also seems to play a role. Less efficient intramolecular enantioselection was observed for the hydrolysis of N-t-BOC-S-metthionine p-nitrophenyl ester with R- and S-enantiomers of [Pd(C₆H₄C*HMeNMe₂)Cl] coordinated to sulfur.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

Reduction of ferricytochrome c by hydrogen atoms: Evidence for intramolecular transfer of reducing equivalent

Direct evidence obtained by means of the technique of pulse radiolysis-kinetic spectrometry, with measurements in the time range 10⁻⁶ to 1 s, is presented that, consequent upon reaction of a single H-atom with a single molecule of ferricytochrome c, a reducing equivalent is transmitted via the protein structure to the ferriheme moiety. Such transmission accounts for at least 70% of the total reduction of the ferri to the ferro state of cytochrome c. The remainder of the total reduction takes place without stages resolvable on the time scale of these experiments. Reduction brought about by H atoms appears to follow a different course than reduction by hydrated electrons. In the latter case, intramolecular transmission of reducing equivalents could not be demonstrated (Lichtin, N. N., Shafferman, A. and Stein, G. (1973) Biochim. Biophys. Acta 314, 117–135).

Not every H-atom reacts with ferricytochrome c at a site which results in conversion of the Fe(III) state to the Fe(II) state. Approximately half of reacting H-atoms do not produce reduction.

The following second order rate constants have been determined in solutions of low ionic strength at 20±2 °C: k[H+ferricytochrome c] = (1.0±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0 and 6.7; k[H+ferrocytochrome c] = (1.3±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0; k[e⁻_aq + ferrocytochrome c] = (1.9±0.4) · 10¹⁰ M⁻¹ · s⁻¹ at pH 6.7.     

Kinetics of some electron-transfer reactions in biological Photosystems II. Study of intramolecular electron-transfer rates between ferredoxin-NADP reductase, NADP radical and oxidized ferredoxin

It has been shown by the pulse radiolysis technique that radiation-generated NADP free radicals (NADP·) first combine with ferredoxin-NADP reductase and then transfer the odd electron by a fast intramolecular process to the enzyme flavin moiety yielding the semiquinone (ferredoxin-NADP reductase, FNR-FADH·). The corresponding first-order rate constant k₁₅ varies with ionic strength from 2.6·10³ s⁻¹ at I = 0.66 M to 2.3·10⁴ s⁻¹ at I = 0.005 M In the presence of ferredoxin-NADP reductase-bound oxidized ferredoxin, the electron cascades, thus further reducing the ferredoxin. The transfer of the electron from the flavin semiquinone (ferredoxin-NADP reductase, FNR-FADH·) to the bound oxidized ferredoxin proceeds at a rate of k₁₈ = 2.36 s⁻¹. This process approaches an equilibrium condition which is in favor of the reverse reaction suggesting that k₋₁₈ > k₁₈.     

Thermostable and alkalophilic maltogenic amylase of Bacillus thermoalkalophilus ET2 in monomer-dimer equilibrium

A gene encoding a thermostable and alkalophilic maltogenic amylase (BTMA) was cloned from the thermophilic bacterium Bacillus thermoalkalophilus ET2. BTMA was composed of 588 amino acids with a predicted molecular mass of 68.8 kDa. The enzyme had an optimal temperature and pH of 70°C and 8, respectively, the highest among maltogenic amylases reported so far. The Tm of BTMA at pH 8 was 76.7°C with an enthalpy of 113.6 kJ mol^-1. Both hydrolysis and transglycosylation activities for various carbohydrates were evident. β-Cyclodextrin (β-CD) and soluble starch were hydrolyzed mainly to maltose, and pullulan to panose. Acarbose, a strong amylase inhibitor, was hydrolyzed by BTMA to glucose and acarviosine-glucose. The K _m and k _cat values of BTMA for β-CD hydrolysis were 0.128 mM and 165.8 s^-1 mM, respectively. The overall catalytic efficiency (k _cat/K _m) of the enzyme was highest toward β-CD. BTMA was present in a monomer-dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer (pH 8.0). This equilibrium could be affected by KCl and enzyme concentrations. The multi-substrate specificity of the enzyme was modulated by the structural differences between monomeric and dimeric forms. Starch was hydrolyzed more readily when monomeric BTMA was prevalent, while the opposite was observed for β-CD.     

Studies on the rate-limiting reaction of photosynthetic oxygen evolution in spinach chloroplasts

The modulated oxygen polarograph has been used to study the rate-determining steps of photosynthetic oxygen evolution in spinach chloroplasts. The rate constant, k, of the reaction has a value of 218±10 (S.E.) s⁻¹ at 23 °C and an activation energy of 7±2 (S.E.) kcal · mol⁻¹. A kinetic isotope experiment indicated that this step is probably not the water-splitting reaction. These findings resemble previous results with the unicellular alga Chlorella (Sinclair, J. and Arnason, T. (1974) Biochim. Biophys. Acta 368, 393–400). In other experiments we changed the pH, O₂ concentration and osmolarity of the medium, and treated the chloroplasts with 1 mM NH₄Cl without detecting any significant change in k. These results suggest that the step is irreversible. However, a significantly lower value of k, 110±20 (S.E.) s⁻¹ was obtained when all salts except 1 mM MgCl₂ were removed from the medium bathing the chloroplasts.     

Irreversible thermal denaturation of uridine phosphorylase from Escherichia coli K-12

Thermal denaturation of uridine phosphorylase from Escherichia coli K-12 has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were obtained at temperature scanning rates of 0.25, 0.5, and 1 K/min. These profiles were analysed using three models of irreversible denaturation which are approximations to the whole Lumry-Eyring model, namely, the one-step model of irreversible denaturation, the Lumry-Eyring model with the fast equilibrating first step, and the model involving two consecutive irreversible steps. In terms of statistics the latter model describes the kinetics of thermal denaturation of uridine phosphorylase more satisfactorily than the two other models. The values of energy activation for the first and second steps calculated for the model involving two consecutive irreversible steps are the following: E_a,1 = 609.3 ± 1.8 kJ/mol and E_a,2 = 446.8 ± 3.2 kJ/mol.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

.     

Kinetics and mechanism of the stoichiometric oxygenation of [Cu(fla)(idpa)]ClO4 [fla=flavonolate, IDPA=3,3′-imino-bis(N,N-dimethylpropylamine)] and the [Cu(fla)(idpa)]ClO4-catalysed oxygenation of flavonol

Oxygenation of [Cu^II(fla)(idpa)]ClO₄ (fla=flavonolate; IDPA=3,3′-iminobis(N,N-dimethylpropylamine)) in dimethylformamide gives [Cu^II(idpa)(O-bs)]ClO₄ (O-bs=O-benzoylsalicylate) and CO. The oxygenolysis of [Cu^II(fla)(idpa)]ClO₄ in DMF was followed by electronic spectroscopy and the rate law −d[{Cu^II(fla)(idpa)}ClO₄]/dt=k_obs[{Cu^II(fla)(idpa)}ClO₄][O₂] was obtained. The rate constant, activation enthalpy and entropy at 373 K are k_obs=6.13±0.16×10⁻³ M⁻¹ s⁻¹, ΔH^‡=64±5 kJ mol⁻¹, ΔS^‡=−120±13 J mol⁻¹ K⁻¹, respectively. The reaction fits a Hammett linear free energy relationship and a higher electron density on copper gives faster oxygenation rates. The complex [Cu^II(fla)(idpa)]ClO₄ has also been found to be a selective catalyst for the oxygenation of flavonol to the corresponding O-benzoylsalicylic acid and CO. The kinetics of the oxygenolysis in DMF was followed by electronic spectroscopy and the following rate law was obtained: −d[flaH]/dt=k_obs[{Cu^II(fla)(idpa)}ClO₄][O₂]. The rate constant, activation enthalpy and entropy at 403 K are k_obs=4.22±0.15×10⁻² M⁻¹ s⁻¹, ΔH^‡=71±6 kJ mol⁻¹, ΔS^‡=−97±15 J mol⁻¹ K⁻¹, respectively.     

Day-night variation of thermoregulatory responses to intraperitoneal electric heating in rats

1. 1.Hypothalamic temperature (T_hy), nonevaporative heat loss (R + C + K), evaporative heat loss (E), thermal conductance (k), metabolic heat production (M) and heat storage (S) of rats were simultaneously measured by direct and indirect calorimetry during internal heat loading (2 W per rat) with an intraperitoneal electric heater.

2. 2.The tests were made twice a day; once during the day (1000–1200 h) and once at night (2200–2400 h) at an ambient temperature of 24°C.

3. 3.The resting values of T_hy, colonic temperature, (R + C + K), E, M and heart rate, and the T_hy threshold for tail skin vasodilation (T_th) during internal heat load were significantly higher at night than during the day.

4. 4.The slopes showing the relationshiop between (R + C + K), k or M and T_hy were significantly steeper during the day than at night after T_hy exceeded T_th.

5. 5.The slopes showing the relationship between E or S and T_hy were not different during the day and at night.

6. 6.These results indicate that the responses of nonevaporative heat loss and heat production to internal heat load vary with the time of day in rats.

Changes in denaturation and rheological properties of collagen-hyaluronic acid scaffolds as a result of temperature dependencies

This report describes the effect of temperature on the mechanical viscoelastic properties such as: storage modulus (E′), loss modulus (E″), and loss tangent (tan δ) of the collagen sponges modified with hyaluronic acid (HA). In order to detect collagen–HA copolymer denaturation and to assess its thermal stability, the differential scanning calorimetry (DSC) supplemented by thermogravimetric (TG) measurements was used. The denaturation temperature (T_d) of unmodified collagen samples increased from 69 to 86 °C for cross-linked samples, respectively. These temperature dependencies show remarkable changes in E′ and E″ at selected temperature up to 226 °C for all samples due to the release of loosely and strongly bound water. The influence of HA on the viscoelastic behavior of collagen is manifested by a shift of the tan δ peak associated with the process of decomposition towards higher temperatures resulting in a higher thermo-stability of the modified scaffolds.     

油蒿资源利用效率在生长季的相对变化及对环境因子的响应

为了探明西北半干旱区典型沙生植物油蒿(Artemisia ordosica)叶水平资源利用效率的相对变化及对环境因子的响应机制, 该研究于2018年5-10月, 使用LI-6400XT便携式光合仪测定了毛乌素沙地油蒿叶片的净光合速率(P_n)、蒸腾速率(E)、叶表面光合有效辐射(PAR_l)、叶表面温度(T_l)、叶表面相对湿度(RH_l), 在实验室计算叶片单位面积氮含量(N_area), 分析了叶片氮利用效率(NUE)、水分利用效率(WUE)、光利用效率(LUE)与环境因子之间的关系及NUE、WUE、LUE之间的相对变化。研究结果表明, 在充足且稳定光强下油蒿的P_n主要受温度的影响, NUE、WUE与VPD_l、T_l之间具有显著负相关关系, NUE、WUE与LUE间为正相关关系, NUE、WUE和LUE最大值分别发生在5、7和9月, 分别为9.43 μmol CO₂·g^-1·s^-1、3.86 mmol·mol^-1、0.04 mol·mol^-1, 资源利用效率的变化主要受P_n的影响。温度通过影响植物N分配来改变P_n, 进而影响着资源利用效率, WUE与LUE显著正相关, 对构建荒漠区生态系统能量交换过程模型有重要意义。     

外源褪黑素与钙离子互作对高温胁迫下黄瓜幼苗过氧化伤害的缓解效应

为了探明褪黑素(MT)和钙离子(Ca²⁺)在调控植物耐热性中是否存在互作关系,以黄瓜幼苗为试材,分析了内源MT和Ca²⁺对高温胁迫的响应;并通过叶面喷施100 μmol·L^-1 MT、10 mmol·L^-1 CaCl₂、3 mmol·L^-1乙二醇二乙醚二胺四乙酸(EGTA,Ca²⁺螯合剂)+100 μmol·L^-1 MT、0.05 mmol·L^-1氯丙嗪(钙调素拮抗剂,CPZ)+100 μmol·L^-1 MT、100 μmol·L^-1氯苯丙氨酸(p-CPA,MT合成抑制剂)+10 mmol·L^-1 CaCl₂和去离子水(H₂O),研究高温下(42/32 ℃)外源MT和Ca²⁺对黄瓜幼苗活性氧积累、抗氧化系统及热激转录因子(HSF)和热激蛋白(HSPs)等的影响。结果表明: 黄瓜幼苗内源MT和Ca²⁺均受高温胁迫诱导;外源MT可上调常温下钙调素蛋白(CaM)、钙依赖蛋白激酶(CDPK5)、钙调磷酸酶B类蛋白(CBL3)、CBL结合蛋白激酶(CIPK2)mRNA表达;CaCl₂处理的MT合成关键基因色氨酸脱羧酶(TDC)、5-羟色胺-N-乙酰转移酶(SNAT)和N-乙酰-5-羟色胺甲基转移酶(ASMT)水平也显著升高,MT含量快速增加。MT和CaCl₂可显著增强高温下黄瓜的抗氧化能力,减少活性氧(ROS)积累,同时上调HSF7、HSP70.1和HSP70.11 mRNA表达,从而减轻高温胁迫引起的过氧化伤害,植株热害症状明显减轻,热害指数和电解质渗漏率显著降低。加入EGTA和CPZ后,MT对黄瓜幼苗抗氧化能力和热激蛋白表达的促进效应明显减弱,Ca²⁺对高温下黄瓜幼苗过氧化伤害的缓解效应也被p-CPA逆转。可见,MT和Ca²⁺均可诱导黄瓜幼苗的耐热性,二者在热胁迫信号转导过程中存在互作关系。     

大气臭氧浓度升高对银杏叶片活性氧代谢及相关基因表达的影响

采用开顶式气室熏蒸法,设置自然条件下臭氧(O₃)浓度(对照,约40 nmol·mol^-1)、80、160及200 nmol·mol^-14个臭氧浓度,观测了不同浓度臭氧条件下银杏叶片可见伤害、活性氧生成量、抗氧化酶活性及相关基因表达变化情况,分析大气臭氧浓度升高对植物活性氧代谢的影响.结果表明: 160和200 nmol·mol^-1 O₃熏蒸明显伤害银杏叶片,80 nmol·mol^-1与对照无差异,无可见伤害.O₃处理20 d后,160和200 nmol·mol^-1条件下银杏叶片的超氧自由基(O₂^-·)产生速率显著高于80 nmol·mol^-1和对照,而80 nmol·mol^-1与对照无差异;O₃处理40 d后,160和200 nmol·mol^-1熏蒸下叶片过氧化氢(H₂O₂)含量显著高于80 nmol·mol^-1和对照,而过氧化氢酶(CAT)活性显著高于80 nmol·mol^-1和对照,各臭氧处理抗坏血酸过氧化物酶(APX)活性均低于对照.熏蒸40 d后,CAT、APX基因的转录表达持续加强;防御素(GbD)的表达强度则随着臭氧浓度的增加及熏蒸时间的延长而呈显著加强.高浓度臭氧胁迫可使银杏叶片活性氧生成量增加、抗氧化酶活性下降、相关基因表达水平上调,有明显可见叶片伤害.     

Comparative thermodynamic elucidation of the structural stability of thermophilic proteins

Differential scanning calorimetry, circular dichroism, and visible absorption spectrophotometry were employed to elucidate the structural stability of thermophilic phycocyanin derived from Cyanidium caldarium, a eucaryotic organism which contains a nucleus, grown in acidic conditions (pH 3.4) at 54°C. The obtained results were compared with those previously reported for thermophilic phycocyanin derived from Synechococcus lividus, a procaryote containing no organized nucleus, grown in alkaline conditions (pH 8.5) at 52°C. The temperature of thermal unfolding (t_d) was found to be comparable between C. caldarium (73°C) and S. lividus (74°C) phycocyanins. The apparent free energy of unfolding (ΔG_[urea]=0) at zero denaturant (urea) concentration was also comparable: 9.1 and 8.7 kcal/mole for unfolding the chromophore part of the protein, and 5.0 and 4.3 kcal/mole for unfolding the apoprotein part of the protein, respectively. These values of t_d and ΔG_[urea]=0 were significantly higher than those previously reported for mesophilic Phormidium luridum phycocyanin (grown at 25°C). These findings revealed that relatively higher values of t_d and ΔG_[urea]=0 were characteristics of thermophilic proteins. In contrast, the enthalpies of completed unfolding (ΔH_d) and the half-completed unfolding (ΔH_d)1/2 for C. caldarium phycocyanin were much lower than those for S. lividus protein (89 versus 180 kcal/mole and 62 versus 115 kcal/mole, respectively). Factors contributing to a lower ΔH_d in C. caldarium protein and the role of charged groups in enhancing the stability of thermophilic proteins were discusse.     

A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

1. Single reduced methyl viologen (MV^.+) acts as an electron donor in a number of enzyme systems. The large changes in extinction coefficient upon oxidation (λ_max 600 nm; MV^.+, = 1.3 · 10⁴ M⁻¹ · cm⁻¹; oxidised form of methyl viologen (MV²⁺), = 0.0) make it ideally suited to kinetic studies of electron transfer reactions using stopped-flow and standard spectrophotometric techniques.

2. A convenient electrochemical preparation of large amounts of MV^.+ has been developed.

3. A commercial stopped-flow apparatus was modified in order to obtain a high degree of anaerobicity.

4. The reaction of MV^.+ with O₂ produced H₂O₂ (k > 5 · 10⁶ M⁻¹ · s⁻¹, pH 7.5, 25 °C). H₂O₂ subsequently reacted with excess MV^.+ (k = 2.3 · 10³ M⁻¹ · s⁻¹, pH 7.5, 25 °C) to produce water. The kinetics of this reaction were complex and have only been interpreted over a limited range of concentrations.

5. The results support the theory that the herbicidal action of methyl viologen (Paraquat, Gramoxone) is due to H₂O₂ (or radicals derived from H₂O₂) induced damage of plant cell membrane.     

Kinetics and equilibria of Ga(III)---thiocyanate complex formation. Mechanism of ligand substitution reactions of Ga(III) in aqueous solution

The kinetics and equilibria of complex formation by Ga(III) with NCS⁻ in aqueous solution have been measured over a range of acidities and temperatures, the contributing paths to the reaction resolved, and their rate constants and activation parameters determined. The hydrolysis equilibria required to carry out this resolution of kinetic behaviour have also been measured.

Unlike the other reported complexation reactions of Ga(III) in aqueous solution, the separate reaction pathways can be assigned with no ambiguity. At 25 °C and ionic strength 0.5 M, the observed forward rate constant for the complex formation is described by {k₁ + k₂K_1h/[H⁺] + k₃K_1hK_2h/[H⁺]²} M⁻¹ s⁻¹. For these conditions, the first and second successive hydrolysis constants of Ga(H₂O)₆³⁺ are given by pK_1h = 3.69 ± 0.01 and pK_2h = 3.74 ± 0.04. The rate constants corresponding to the reactions of the species Ga(H₂O)₆³⁺, Ga(H₂O)₅(OH)²⁺ and Ga(H₂O)₄(OH)₂⁺ with NCS⁻ are k₁ = 57 ± 4 M^{−1 −1}, k₂ = (1.08 ± 0.01) × 10⁵ M⁻¹ s⁻¹ and k₃ = 3 × 10⁶ M⁻¹ s⁻¹ respectively. The complexation equilibrium quotient [GaNCS²⁺]/([Ga³⁺][NCS⁻]) has been independently determined by spectrophotometric titration to be 20.8 ± 0.3 M⁻¹ at 25 °C and ionic strength 0.5 M.

These kinetic results lead to an interpretation of the data, and a reinterpretation of other data for aquo-Ga(III) complex formation kinetics from the literature which support the assignment of a dissociative interchange mechanism for these reactions rather than the associative activation mode sometimes proposed.