OXYGEN RADICALS MEDIATE CELL INACTIVATION BY ACRIDINE DYES, FLUORESCEIN, and LUCIFER YELLOW CH

Acridine dyes, fluorescein and lucifer yellow CH are fluorescent photosensitizers used experimentally to selectively stain and photodynamically destroy eukaryotic cells and subcellular structures. We have determined that the mechanism of light- and oxygen-dependent inactivation of E. coli by these dyes involves oxygen radicals and hydrogen peroxide. All of the dyes oxidized NAD(P)H+ under illumination. Superoxide (O2), detected as the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c, was a major product of the dye sensitized photooxidation. Cationic acridine dyes penetrated the membranes of E. coli and were photoreduced intracellularly. Reduced dyes diffused back into the medium and mediated the reduction of extracellular ferricytochrome c. The anionic dyes fluorescein and lucifer yellow CH were unable to mediate extracellular cytochrome c reduction, indicating that these dyes were impermeable to the E. coli membrane. Acridine dyes, when illuminated, inhibited the growth of E. coli in a rich medium, and induced the synthesis of SOD. Fluorescein and lucifer yellow CH did not inhibit growth or induce SOD synthesis because they were unable to enter the cells. Superoxide (O2) and hydrogen peroxide (H2O2), generated by the enzyme xanthine oxidase were toxic to E. coli B. Inactivation by xanthine oxidase was partially inhibited by exogenous SOD and completely inhibited by exogenous catalase or SOD plus catalase. Similarly, exogenous SOD plus catalase protected against inactivation by acridines and fluorescein-NADH or lucifer yellow CH-NADH mixtures. Prior induction of superoxide dismutase and catalase in E. coli B significantly protected cells against a subsequent challenge by illuminated acridine dyes. SOD and catalases preinduction combined with additions of exogenous SOD and catalase completely protected E. coli B against photodynamic inactivation by acridine yellow. The hydroxyl radical scavengers, dimethyl sulfoxide, sodium benzoate and thiourea, protected E. coli B against photodynamic inactivation by acridine orange. The results implicate O2, H2O2, and the hydroxyl radical (OH) as underlying molecular agents of the phototoxicity mediated by acridine orange, acridine yellow, fluorescein and lucifer yellow CH.     

碱性鲁米诺体系在玻碳和铂电极上的电位分辨电致化学发光研究

提出一种使用循环伏安（ＣＶ）扫描电位下的电致化学发光（ＥＣＬ）研究方法,在自制的仪器系统中同时进行ｉ－Ｅ和Ｉ－Ｅ测量,获得对应的ＣＶ和电位分辨的电致化学发光（ＰＲＥＣＬ）曲线。首先发现碱性鲁米诺体系在玻碳电极（ＧＣＥ）上呈现２个阳极发光峰（在０．３２Ｖ、０．３９Ｖｖｓ．Ａｇ）和１个阴极发光峰（在－０．６２Ｖ）,在Ｐｔ电极上呈现２个阳极发光峰（在０．４９Ｖ、０．７５Ｖ）。结合ＣＶ、一阶微分伏安、Ｃｌ     

人血多形核白细胞呼吸爆发中活性氧与活性氧模拟酶的关系研究

用多形核白细胞－鲁米诺化学发光法测定具有超氧化物歧化酶（ＳＯＤ）和过氧化氢酶（ＣＡＴ）双功能模拟酶－金属卟啉（四磺酸基苯基铁卟啉、四磺酸基苯基铜卟啉、四磺酸基苯基锰卟啉、四磺酸基苯基钴卟啉）对呼吸爆发所产生的活性氧的影响,结果表明,模拟酶对爆发反应中细胞发光产生抑制,即对活性氧有清除作用;且同一物质随着浓度由１０＾－７－１０＾－５ｍｏｌ／Ｌ,上升而增强。     

Direct determination of horseradish peroxidase encapsulated in liposomes by using luminol chemiluminescence.

Horseradish peroxidase (HRP) encapsulated in liposomes was directly detected by using luminol chemiluminescence (CL) with H2O2 without lysis of liposomes. At a low concentration of H2O2, the initial rate of HRP-catalyzed luminol CL in liposomes was slower than that of HRP-catalyzed luminol CL in a lipid-free bulk solution. The decrease in the initial rate of the CL reaction in liposomes was due to the membrane permeation of luminol and H2O2. At a high concentration of H2O2, the initial rate of the CL reaction in liposomes was the same as that in a lipid-free bulk solution. The CL measurement conditions in both a lipid-free bulk solution and in liposomes were optimized in the concentrations of luminol and H2O2 by measuring the CL response curves, in which only one peak appeared and the CL intensity was maximal. The CL intensity observed in HRP-catalyzed luminol CL in liposomes was a factor of seven greater than that observed in a lipid-free bulk solution. The CL intensity was dependent on the amount of HRP-encapsulated liposomes used. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 3 compared with that in HRP-catalyzed luminol CL in a lipid-free bulk solution.     

A chemiluminescence method for the detection of electrochemically generated H2O2 and ferryl porphyrin

Electrochemical formation of H2O2 and the subsequent ferryl porphyrin were examined by measuring luminol chemiluminescence and absorption spectrum using flow-injection method. Emission was observed under the cathodic potential (0.05 V at pH 2.0 and -0.3 V at pH 11.0) by the electrochemical reduction of buffer electrolytes solution but no emission was observed at anodic potentials. Fe(III)TMPyP solution was added at the down stream of the working electrode and was essential for the emission. Removal of dissolved O2 resulted in the decrease of emission intensity by more than 70%. In order to examine the lifetime of reduced active species, delay tubes were used in between working electrode and Fe(III)TMPyP inlet. Experimental results suggested the active species were stable for quite long. The emission was quenched considerably (>90%) when hydroperoxy catalase was added at the down stream of the working electrode whereas SOD had little effect. Significant inhibition of the emission by the addition of alkene at the down stream of the Fe(III)TMPyP inlet was considered as evidence of oxo-ferryl formation. The spectra at reduction potential under aerated condition were shifted to the longer wavelength (>430 nm) compared to the original spectrum of Fe(III)TMPyP (422 nm). All the spectra were perfectly reproduced by a combination of Fe(III)TMPyP and O=Fe(IV)TMPyP (438 nm) spectra. These observations lead to the conclusion that H2O2 was produced first by electrochemical reduction of O2, which then converted Fe(III)TMPyP into O=Fe(IV)TMPyP to activate luminol. The current efficiencies for the formation of H2O2 were estimated as about 30-65% in all over the pH.     

牛磺酸对大乳鼠心肌细胞抗氧化作用的荧光免疫化学分析

应用免疫和化学分析方法探讨牛磺酸(Taurine,Tau)对原代培养的新生大乳鼠心肌细胞的保护作用,为心血管疾病的治疗提供理论依据.将原代培养的乳鼠心肌细胞随机分为对照组、过氧化氢(H2O2)损伤模型组以及H2O2损伤加药物治疗组.预先12h给予Tau治疗药物后,使用H2O2损伤心肌细胞4h,在倒置显微镜观察Tau对心肌细胞形态学的影响,3-(4,5-二甲基噻唑-2Y-2,5-二苯基四氮唑溴盐(3-(4,5) - Dimethylthiahiazo(-z-y1)-3,5- diphenytetrazoliumromide,MTT)化学分析法测定心肌细胞的存活率,化学试剂盒检测心肌细胞内超氧化物歧化酶(Superoxide dismutase,SOD)活性及丙二醛(Malondialdehyde,MDA)含量的变化,利用荧光探针DCFH DA分析检测心肌细胞中活性氧的水平.结果表明,H2 O2损伤心肌细胞4h后,Tau可使吸光度升高,并增加心肌细胞SOD活性、降低MDA含量及活性氧水平下降.由此可见,采用荧光免疫分析方法可证实Tau能减轻自由基对心肌细胞的损伤,从而达到保护心脏的作用.     

Development and validation of a screening procedure for the assessment of inhibition using a recombinant enzyme

Myeloperoxidase (MPO, E.C. 1.1.11.7) is a heme-containing enzyme that catalyses the synthesis of hypochlorous acid (HOCl) in the presence of hydrogen peroxide (H2O2) and chlorine anions. The production of HOCl is kept under strict control of neutrophils. However, in several pathological conditions, MPO is leaked in the extracellular fluid, which involves an over-production of reactive oxygen species like HOCl and promotes the damages caused by neutrophils. As a consequence, the inhibition of MPO by various agents has been investigated and a variety of molecules have been evaluated for this activity in different models. The present study aims to describe and validate a rapid screening method based on the taurine assay and using a recombinant MPO. After validation of the stock solutions used during the experiments, the amount of MPO for the completion of the reaction was measured and fixed to an optimal value. The inhibiting concentration at 50% of flufenamic acid (taken as a reference molecule) was then assessed in both a simple tube test and a microplate test and delivered similar results (1.3+/-0.2 microM vs 1.4+/-0.2 microM, P=0.2). Finally, different molecules able to inhibit MPO were evaluated in this rapid assay system providing results comparable to literature. The high throughput screening is undoubtedly a first line assessment method which affords the selection of inhibitors and permits to reduce the number of candidates for a further elucidation of the mechanism of MPO inhibition.     

Existence of a new reactive intermediate oxygen species in hypoxanthine and xanthine oxidase reaction

We investigated a hypoxanthine (HPX) and xanthine oxidase (XOD) reaction by using a luminol analog 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-mediated chemiluminescence (CL) response. Addition of a high activity of superoxide dismutase (SOD), a potent O2* scavenger, and of a high concentration of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a potent spin trapping agent, diminished completely the CL response. Whereas a high concentration of dimethyl sulfoxide (DMSO), as a potent *OH scavenger could not attain to the complete diminishment of the CL response. It has been reported that luminol monoanion reacts with *OH to form luminol radical, and then resultant luminol radical reacts with O2* to elicit CL response. Complete scavenging for *OH is assumed to result in lack of luminol radical, which in turn induces lack of CL response. However, our results did not support the idea. Furthermore, we examined the effect of L-012 on the DMPO-OOH formation in the presence or absence of DMSO in the HPX-XOD system by applying an electron spin resonance (ESR)-spin trapping method. The DMPO-OOH formation was inhibited even in the presence of DMSO, and the rate constant (k2) between L-012 and O2* obtained in the presence of DMSO was 9.77 x 10(2) M(-1) s(-1) and the constant in the absence of DMSO was 2.97 x 10(3) M(-1) s(-1). The data suggests that L-012 is converted to a radical form that reacts with O2* even under the conditions of the absence of *OH. From these, we postulate that the existence of a reactive intermediate oxygen species in the HPX-XOD system.     

Inhibition of superoxide dismutase, Vitamin C and glutathione on chemiluminescence produced by luminol and the mixture of sulfite and bisulfite

In a system which consisted of luminol (3-aminophthalhydrazide), cobalt sulfate (CoSO4), alkaline buffer and the mixture of NaSO3 and sodium bisulfite (NaHSO3) (sulfite and bisulfite=3:1, m/m), a strong chemiluminescence (CL) was observed using a BPCL ultra-weak luminometer. The CL signals resulted from 3-aminophthalate (the product of oxidized luminol), and were affected by the buffer pH, buffer medium and the concentrations of luminol, CoSO4 and the NaSO3-NaHSO3 mixture. The observation that the CL intensities were inhibited by superoxide dismutase (SOD), Vitamin C (Vc) and glutathione (GSH) in a dose-dependent manner suggested that superoxide radical (O2*-) was involved in the CL reaction and responsible for oxidation of luminol.     

Luminol-Based Chemiluminescent Signals: Clinical and Non-clinical Application and Future Uses

Chemiluminescence (CL) is an important method for quantification and analysis of various macromolecules. A wide range of CL agents such as luminol, hydrogen peroxide, fluorescein, dioxetanes and derivatives of oxalate, and acridinium dyes are used according to their biological specificity and utility. This review describes the application of luminol chemiluminescence (LCL) in forensic, biomedical, and clinical sciences. LCL is a very useful detection method due to its selectivity, simplicity, low cost, and high sensitivity. LCL has a dynamic range of applications, including quantification and detection of macro and micromolecules such as proteins, carbohydrates, DNA, and RNA. Luminol-based methods are used in environmental monitoring as biosensors, in the pharmaceutical industry for cellular localization and as biological tracers, and in reporter gene-based assays and several other immunoassays. Here, we also provide information about different compounds that may enhance or inhibit the LCL along with the effect of pH and concentration on LCL. This review covers most of the significant information related to the applications of luminol in different fields.     

Heparan sulfate degradation via reductive homolysis of its N-chloro derivatives

The highly basic heme enzyme myeloperoxidase (MPO), which is released by activated phagocytes, catalyzes the production of the potent oxidant hypochlorite (HOCl) from H(2)O(2) and chloride ions (Cl(-)). Heparan sulfate proteoglycans are key components of the extracellular matrix and cell surfaces and are known to bind MPO avidly via their negatively charged heparan sulfate chains. Reaction of heparan sulfate with HOCl generates polymer-derived N-chloro derivatives (chloramines, dichloramines, N-chlorosulfonamides, and chloramides). In this study, it is shown that heparan sulfate N-chloro derivatives are decomposed in the presence of redox-active transition-metal ions and superoxide (O(2)(*-)). These processes initiate polymer modification/fragmentation. Radical intermediates in these processes have been identified by EPR spectroscopy and spin trapping. Evidence has been obtained that the N-chloro derivatives undergo reductive homolysis to nitrogen-centered (aminyl, N-chloroaminyl, sulfonamidyl, and amidyl) radicals that generate carbon-centered radicals via rapid, intramolecular hydrogen atom abstraction reactions (1,2- and/or 1,5-shifts). In the case of the sulfonamidyl radicals, rearrangement via 1,2-shifts and beta-scission of the resultant C-2 carbon-centered radicals to yield SO(3)(*-) and C-2 imines is near quantitative based on the yield of SO(4)(2-), the decomposition product of SO(3)(*-). The formation of strand breaks and chromophores during these reactions is attributed to the formation and subsequent heterolytic rearrangement of the C-2 imines. The degradation of heparan sulfate via reductive homolysis of its N-chloro derivatives may be of significance at sites of inflammation, where MPO-derived HOCl is produced in high concentration and transition-metal ions and O(2)(*-) are known to be present or generated.     

Synthesis, characterization, and electrochemiluminescence of luminol-reduced gold nanoparticles and their application in a hydrogen peroxide sensor

It was found that chloroauric acid (HAuCl(4)) could be directly reduced by the luminescent reagent luminol in aqueous solution to form gold nanoparticles (AuNPs), the size of which depended on the amount of luminol. The morphology and surface state of as-prepared AuNPs were characterized by transmission electron microscopy, UV/visible spectroscopy, X-ray photoelectron spectroscopy, FTIR spectroscopy, and thermogravimetric analysis. All results indicated that residual luminol and its oxidation product 3-aminophthalate coexisted on the surface of AuNPs through the weak covalent interaction between gold and nitrogen atoms in their amino groups. Subsequently, a luminol-capped AuNP-modified electrode was fabricated by the immobilization of AuNPs on a gold electrode by virtue of cysteine molecules and then immersion in a luminol solution. The modified electrode was characterized by cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy. The as-prepared modified electrode exhibited an electrochemiluminescence (ECL) response in alkaline aqueous solution under a double-step potential. H2O2 was found to enhance the ECL. On this basis, an ECL sensor for the detection of H2O2 was developed. The method is simple, fast, and reagent free. It is applicable to the determination of H2O2 in the range of 3x10(-7)-1x10(-3) mol L(-1) with a detection limit of 1x10(-7) mol L(-1) (S/N=3).     

Bactericidal mechanism of Ag/Al2O3 against Escherichia coli

The bactericidal process of Ag/Al2O3 to Escherichia coli has been investigated to clarify the bactericidal mechanism. In SEM images, the configuration of E. coli cells contacting with the catalyst surface was quite different from that contacting with AgNO3 solution, which indicated that the Ag+ eluted from the catalyst did not play an important role in the bactericidal process. The bactericidal experiments strongly confirmed the contribution of multiform reactive oxygen species (ROS) (super oxide dismutase (SOD) and catalase as the scavengers for O2*- and H2O2, respectively) to bactericidal effect on the catalyst surface. Furthermore, the surface modification of Ag/Al2O3 by ultraviolet and formaldehyde influenced the bactericidal effect obviously, which not only confirmed the bactericidal mechanism of catalytic oxidation but also provided evidence for the synergistic effect between Ag and Al2O3 on the catalyst surface.     

电化学发光淬灭法测定SOD

在硼酸缓冲溶液（pH6．7）中性介质中,O2和H2O2均对鲁米诺的电化学发光（ECL）有敏化作用,而超氧化物歧化酶（SOD）对此敏化的电化学发光具有淬灭作用,SOD的浓度与两种发光体系的ECL光强成线性关系,可用于SOD的测定,为SOD的测定提供了一种灵敏、可靠的测定方法。     

Prosthetic heme modification during halide ion oxidation. Demonstration of chloride oxidation by horseradish peroxidase

Myeloperoxidase (MPO), eosinophil peroxidase (EPO), and chloroperoxidase can oxidize iodide, bromide, and chloride, but most peroxidases, including the prototypical horseradish peroxidase (HRP), reportedly only oxidize iodide and, in some cases, bromide. We report here that incubation of HRP with Br(-) and H(2)O(2) at acidic pH results in both bromination of monochlorodimedone and modification of the heme group. Mass spectrometry indicates that the heme 2- and 4-vinyl groups are modified by either replacement of a vinyl hydrogen by a bromide or addition of HOBr to give a bromohydrin. These reactions do not occur if protein-free heme and Br(-) are co-incubated with H(2)O(2) or if the HRP reaction is carried out at pH 7. Surprisingly, similar prosthetic heme modifications occur in incubations of HRP with H(2)O(2) and Cl(-). A mechanism is proposed involving oxidation of Br(-) or Cl(-) to give HOBr or HOCl, respectively, followed by addition to a vinyl group. In the reaction with Cl(-), a meso-chloro heme adduct is also formed. This first demonstration of Cl(-) oxidation by HRP, and the finding that prosthetic heme modification occurs when Br(-) or Cl(-) is oxidized in the absence of a cosubstrate, show that only modest tuning is required to achieve the unique chloride oxidation activity of MPO and EPO. The results raise the question of how the prosthetic hemes of MPO and EPO, whose function is to produce oxidized halide species, escape modification.     

Protecting peroxidase activity of multilayer enzyme-polyion films using outer catalase layers

Films constructed layer-by-layer on electrodes with architecture {protein/hyaluronic acid (HA)}n containing myoglobin (Mb) or horseradish peroxidase (HRP) were protected against protein damage by H2O2 by using outer catalase layers. Peroxidase activity for substrate oxidation requires activation by H2O2, but {protein/HA}n films without outer catalase layers are damaged slowly and irreversibly by H2O2. The rate and extent of damage were decreased dramatically by adding outer catalase layers to decompose H2O2. Comparative studies suggest that protection results from catalase decomposing a fraction of the H2O2 as it enters the film, rather than by an in-film diffusion barrier. The outer catalase layers controlled the rate of H2O2 entry into inner regions of the film, and they biased the system to favor electrocatalytic peroxide reduction over enzyme damage. Catalase-protected {protein/HA}n films had an increased linear concentration range for H2O2 detection. This approach offers an effective way to protect biosensors from damage by H2O2.     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

SANGUINARINE, A PHOTOTOXIC H2O2-PRODUCING ALKALOID

Sanguinarine chloride, a quaternary salt of a benzophenanthrene alkaloid, was phototoxic to catalase-deficient strains of Escherichia coli but not to Trichoplusia ni (cabbage looper moth larvae), an insect with high levels of catalase activity. Chemical analyses confirm that sanguinarine is an efficient producer of H2O2. This differential toxicity suggests that the mode of phototoxic action involves production of H2O2 which could be detoxified in many organisms by catalase.     

The enhanced electrochemiluminescence of luminol on the nickel phthalocyanine modified electrode

A glassy carbon electrode (GCE) modified with nickel(II) tetrasulfophthalocyanine (NiTSPc) and Nafion was used for the investigation of the catalytic oxidation of luminol. The modified electrode was found to much more effectively improve the emission of electrochemiluminescence(ECL) of luminol in a solution containing hydrogen peroxide. The enhanced ECL signal corresponded to the catalytic oxidation of both luminol and H(2)O(2) by NiTSPc. Attached Ni(II) on GCE was oxidised to Ni(III) and then used as the catalyst for the chemiluminescence of luminol. The enhanced stability of the ECL signal with Nafion would mainly result from the prevention of the dissolution of NiTSPc and the adsorption of the oxidation product of luminol on the electrode surface. The proposed method enables a detection limit for luminal of 6.0 x 10(-8) mol L(-1) to be achieved in the presence of H(2)O(2) in the neutral solution. The enhanced ECL intensity had a linear relationship with the concentration of luminol in the range of 1.0 x 10(-7)-8.0 x 10(-6) mol L(-1).     

Toxicities of dicyanobenzofurazans with formation of superoxide in Escherichia coli

The toxicities of some benzofurazans (BZs), benzofurazan (1), 4,7-dimethylbenzofurazan (2), 4,7-dibromobenzofurazan (3), 4-bromo-6-cyanobenzofurazan (4), 4,7-dicyanobenzofurazan (5) and 4,5-dicyanobenzofurazan (6), were examined on Escherichia coli. Compound 5 at 4 microM and compound 6 at 7 microM completely inhibited the growth of E. coli in a simple nutritionally restricted medium (GM medium). These compounds were more toxic in GM medium than in a nutritionally rich medium (YE medium), which contained yeast extract as an additive in GM medium. Compound 4 also inhibited the growth of E. coli at 300 microM in GM medium. The toxicities of BZs were in the order of 1 approximately 2 approximately 3 less than 4 approximately 5 approximately 6. Compounds 4, 5 and 6 induced manganese-superoxide dismutase (Mn-SOD) and catalase activities of E. coli in YE medium. The induced SOD and catalase provide a defense against the potential cytotoxicities of O2- and H2O2. The rate of dioxygen uptake in cyanide-resistant respiration of E. coli was dependent on the concentration of 5, and was correlated with the induction of SOD and catalase. The reduction potentials of BZs followed the order of 1 approximately 2 less than 3 less than 4 less than 5 approximately 6. Compounds 5 and 6, which had redox potentials higher than those of the other BZs, are thought to be more readily reduced in the living system.(ABSTRACT TRUNCATED AT 250 WORDS)