Scavenging of free radicals in gas-phase mainstream cigarette smoke by immobilized catalase at filter level

Catalase is well known as capable of inducing the decomposition of H₂O₂. In this study, a kind of immobilized catalase (entrapped in cross-linked chitosan beads) was dispersed in conventional acetate filter as an antioxidant additive. Quantitative estimation of the free radicals in mainstream cigarette smoke (MCS) was performed to address the effect of this modified filter. It was found that the levels of PBN adduct and NO^?/NO₂^? associated with the gas-phase mainstream cigarette smoke (GPCS) were efficiently decreased by ～40% through catalase filtering. Besides, the modified filter was found to lower the MCS-induced adverse biological effects including lipid peroxidation and mutagenicity. This was proved to be substantially attributed to the catalase-dependent breakdown of NO^?, which was stimulated by some of peroxides (most probably being H₂O₂), the dismutation products of tar particulate matters (TPM). These results highlighted a promising approach to reduce the smoking-associated health risks to passive smokers. Moreover, the mechanisms of catalase filtering may be helpful for the development of appropriate immobilized enzyme systems to be applied for reducing health risks associated with gaseous pollutants.     

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

Activity of magnetite-immobilized catalase in hydrogen peroxide decomposition

The present work analyzes the activity in decomposition of H₂O₂ using magnetite-immobilized catalase. The support of catalase is a glutaraldehyde-treated magnetite (Fe₃O₄). The data obtained in the H₂O₂ decomposition are analyzed. The fitting of the initial rate of the H₂O₂ decomposition versus hydrogen peroxide concentration data is discussed using a specific program for enzyme kinetics modeling (Leonora). The free catalase from Aspergillus niger (3.5 or 10 U/mL) does not show substrate inactivation up to 0.4 M H₂O₂. The immobilized catalase at low catalyst concentration shows substrate inhibition. Using 1 mg/mL of supported catalase the predicted maximum activity is higher than in the case of the free catalase at similar catalase concentration, although the optimum temperature is lower (40 °C versus 60 °C).     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

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.     

Inactivation of Yeast Glutathione Reductase by Fenton Systems: Effect of Metal Chelators, Catecholamines and Thiol Compounds

Oxygen radical generating systems, namely, Cu(II)/ H₂O₂, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H₂O₂/catecholamine and Cu(II)/H₂O₂/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H₂O₂ enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H₂O₂ depended on Cu(II) and H₂O₂ concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO^˙ scavengers prevented GR inactivation by Cu(II)/H₂O₂ and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropi-onylglycine and penicillamine enhanced the effect of Cu(II)/H₂O₂ in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H₂O₂ effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and try-panothione disulfide effectively protected GR against Cu(II)/H₂O₂ inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS*⁺. GR inactivation by the systems assayed correlated with their capability for HO* radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.     

H2O2 and Src-dependent transactivation of the EGF receptor mediates the stimulatory effect of leptin on renal ERK and Na+, K+-ATPase

We examined the mechanism through which leptin increases Na⁺, K⁺-ATPase activity in the rat kidney. Leptin was infused under anaesthesia into the abdominal aorta proximally to the renal arteries and then Na⁺, K⁺-ATPase activity was measured in the renal cortex and medulla. Leptin (1 μg/kg min) increased Na⁺, K⁺-ATPase activity after 3 h of infusion, which was accompanied by the increase in urinary H₂O₂ excretion and phosphorylation level of extracellular signal regulated kinase (ERK). The effect of leptin on ERK and Na⁺, K⁺-ATPase was abolished by catalase, specific inhibitors of epidermal growth factor (EGF) receptor, AG1478 and PD158780, as well as by ERK inhibitor, PD98059, and was mimicked by both exogenous H₂O₂ and EGF. The effect of leptin was also prevented by the inhibitor of Src tyrosine kinase, PP2. Leptin and H₂O₂ increased Src phosphorylation at Tyr⁴¹⁸. We conclude that leptin-induced stimulation of renal Na⁺, K⁺-ATPase involves H₂O₂ generation, Src kinase, transactivation of the EGF receptor, and stimulation of ERK.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

Phenotypic analysis of the ccp1Delta and ccp1Delta-ccp1W191F mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

Lethality of hydrogen peroxide in wild type and superoxide dismutase mutants of Escherichia coli. (A hypothesis on the mechanism of H2O2-induced inactivation of Escherichia coli)

The toxicity of H₂O₂ in Escherichia coli wild type and superoxide dismutase mutants was investigated under different experimental conditions. Cells were either grown aerobically, and then treated in M9 salts or K medium, or grown anoxically, and then treated in K medium. Results have demonstrated that the wild type and superoxide dismutase mutants display a markedly different sensitivity to both modes of lethality produced by H₂O₂ (i.e. mode one killing, which is produced by concentrations of H₂O₂ lower than 5 mM, and mode two killing which results from the insult generated by concentrations of H₂O₂ higher than 10 mM). Although the data obtained do not clarify the molecular basis of H₂O₂ toxicity and/or do not explain the specific function of superoxide ions in H₂O₂-induced bacterial inactivation, they certainly demonstrate that the latter species plays a key role in both modes of H₂O₂ lethality. A mechanism of H₂O₂ toxicity in E. coli is proposed, involving the action of a hypothetical enzyme which should work as an O₂^-• generating system. This enzyme should be active at low concentrations of H₂O₂ (<5 mM) and high concentrations of the oxidant (>5 mM) should inactivate the same enzyme. Superoxide ions would then be produced and result in mode one lethality. The resistance at intermediate H₂O₂ concentrations may be dependent on the inactivation of such enzyme with no superoxide ions being produced at levels of H₂O₂ in the range 5–10 mM. Mode two killing could be produced by the hydroxyl radical in concert with superoxide ions, chemically produced via the reaction of high concentrations of H₂O₂ (>10 mM) with hydroxyl radicals. The rate of hydroxyl radical production may be increased by the higher availability of Fe²⁺ since superoxide ions may also reduce trivalent iron to the divalent form.     

Oxidant-mediated AA release from astrocytes involves cPLA(2) and iPLA(2)

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H₂O₂ have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H₂O₂ and menadione, a compound known to release H₂O₂ intracellularly, were used to examine the phospholipases A₂ (PLA₂) responsible for AA release from primary murine astrocytes. Both H₂O₂ and menadione dose-dependently stimulated AA release, and the release mediated by H₂O₂ was completely inhibited by catalase. H₂O₂ also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A₂ (cPLA₂). However, complete inhibition of cPLA₂ phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H₂O₂-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA₂ and the Ca²⁺-independent iPLA₂, nearly completely inhibited H₂O₂-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA₂, only inhibited H₂O₂-mediated AA release by 40%. Along with the observation that H₂O₂-mediated AA release was only partially inhibited upon chelating intracellular Ca²⁺ by BAPTA, these results indicate the involvement of both cPLA₂ and iPLA₂ in H₂O₂-mediated AA release in murine astrocytes.     

Ca2+参与H2O2促进盐碱混合胁迫下燕麦种子的萌发和成苗

盐碱胁迫是制约作物高产优质的重要因素,Ca²⁺和H₂O₂作为信号分子参与作物逆境响应调节。为了解Ca²⁺是否参与H₂O₂对盐碱胁迫下植物种子萌发和成苗的调控,以燕麦（Avena nude）为试验材料,采用隶属函数分析方法,研究了胞外游离Ca²⁺螯合剂EGTA、质膜Ca²⁺通道阻断剂LaCl₃和液泡膜Ca²⁺释放抑制剂钌红（RR）与H₂O₂共处理对盐碱混合（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）胁迫下种子萌发和成苗的影响。结果表明,25~200 mmol·L^-1盐碱混合胁迫显著抑制燕麦的种子萌发和成苗,抑制程度随浓度提高而增强;0.001~2 mmol·L^-1 H₂O₂能够促进燕麦种子的萌发和成苗,且0.5 mmol·L^-1 H₂O₂可以显著缓解75 mmol·L^-1盐碱混合胁迫对燕麦种子萌发和成苗的抑制作用;而EGTA、LaCl₃和RR均能消减H₂O₂对盐碱混合胁迫下燕麦种子萌发和成苗的促进作用。表明Ca²⁺参与H₂O₂促进盐碱混合胁迫下燕麦种子萌发和成苗的信号转导过程。     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

胞外H2O2及NADPH氧化酶参与了铜胁迫对植物细胞死亡的诱导

以烟草悬浮细胞BY-2(Nicotiana tabacum L.cv.Bright Yellow-2)为材料,探讨了在铜离子胁迫下植物细胞死亡发生过程中胞外H₂O₂及NADPH氧化酶所扮演的角色。实验结果表明,随着外源CuCl₂浓度的上升(从0~700 μmol·L^-1),细胞死亡水平不断上升,且胞外H₂O₂的水平也不断增加。在300 μmol·L^-1的CuCl₂诱导细胞死亡的过程中,加入H₂O₂清除剂N-N-二甲基硫脲(DMTU)降低了胞外CuCl₂胁迫下H₂O₂含量增加的同时也降低了细胞死亡水平的上升,这一观察表明了铜离子胁迫所导致的细胞死亡的发生和胞外H₂O₂的增加有关。进一步的研究表明,300 μmol·L^-1 CuCl₂的胁迫导致了NADPH氧化酶活性的显著性上升,而加入NADPH氧化酶的抑制剂（二亚苯基碘,DPI,)则降低了CuCl₂胁迫所导致的细胞死亡和胞外H₂O₂含量的上升。上述结果表明,胞外H₂O₂和NADPH氧化酶参与了CuCl₂对植物细胞死亡的诱导作用。     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

外源H2O2对盐碱混合胁迫下裸燕麦幼苗生长和抗性生理的影响

为探讨信号分子过氧化氢（H₂O₂）增强裸燕麦盐碱耐性的作用及其生理机制,以裸燕麦品种‘定莜6号’为材料,在日光温室内用珍珠岩培养幼苗至三叶一心期时叶面喷施0.01 mmol·L^-1 H₂O₂的同时根部浇灌75 mmol·L^-1盐碱混合溶液（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）或添加H₂O₂淬灭剂二甲基硫脲（DMTU）,研究对幼苗生长及叶片光合色素含量、活性氧代谢和渗透调节物质积累的影响。结果表明：喷施H₂O₂能够缓解盐碱混合胁迫对裸燕麦幼苗生长的抑制,提高幼苗根长、株高和植株干重及叶片叶绿素a、叶绿素b、总叶绿素、类胡萝卜素含量和超氧化物歧化酶、过氧化物酶、过氧化氢酶、抗坏血酸过氧化物酶活性,降低超氧阴离子、H₂O₂、丙二醛、抗坏血酸、谷胱甘肽和游离氨基酸含量,促进抗氧化物质类黄酮、总酚和原花青素及渗透调节物质可溶性蛋白质、可溶性糖和脯氨酸积累。添加DMTU部分或完全逆转了H₂O₂的上述作用。采用隶属函数综合评价显示,喷施H₂O₂提高了盐碱混合胁迫下裸燕麦幼苗的综合评价值D,添加DMTU完全逆转了H₂O₂对D值的提升作用。表明外源H₂O₂通过参与活性氧代谢和渗透调节物质积累等生理代谢调控缓解盐碱混合胁迫诱导的氧化伤害和生长抑制,从而提高裸燕麦对盐碱胁迫的适应能力。     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

过氧化氢缓解裸燕麦幼苗低温胁迫的主成分和隶属函数分析

为探明信号分子过氧化氢（H₂O₂）提高裸燕麦幼苗耐冷性的作用,以‘定莜6号’沙培幼苗为材料,在3叶期喷施10 μmol·L^-1 H₂O₂ 12 h后于8℃/5℃（昼/夜）条件下低温胁迫,以喷蒸馏水（H₂O）为对照,分别在低温处理的0、1、2、3、4、5 d取幼苗叶片测定超氧阴离子（O₂^-·）、H₂O₂、丙二醛（MDA）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）、抗坏血酸过氧化物酶（APX）、抗坏血酸（AsA）、谷胱甘肽（GSH）、可溶性糖（SS）、脯氨酸（Pro）、可溶性蛋白质（SP）和热稳定性蛋白质（HSP）13项与耐冷性有关的生理指标及低温处理5 d后植株株高和生物量增量,采用主成分和隶属函数分析综合评价H₂O₂对裸燕麦幼苗耐冷性的影响。结果表明,与对照相比,喷施H₂O₂显著提高了低温胁迫下裸燕麦幼苗株高和生物量增量,降低了裸燕麦幼苗叶片O₂^-·和MDA及低温胁迫2~5 d的H₂O₂含量,促进低温胁迫期间裸燕麦幼苗叶片SOD、CAT、POD和APX活性提高及AsA、GSH、SS、Pro、SP和HSP积累。主成分分析13项生理指标离差标准化数据,提取的前4个主成分累积方差贡献率达85.6%;隶属函数综合评价4个主成分得分值显示,喷施H₂O₂显著提高了低温胁迫0~5 d的综合评价值。表明喷施H₂O₂能够通过调控生理生化代谢提高裸燕麦幼苗的耐冷性。     

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.