Spectrofluorometric analysis of hydrogen peroxide

The pH of maximum fluorescence (above pH 7) and the optimal excitation and emission wavelengths (468 nm and 519 nm, respectively) were determined for 2′,7′-dichlorofluorescein (DCF). The stoichiometry after hydrolysis of the oxidation of the stable nonfluorescent compound 2′,7′-dichlorofluorescin diacetate (LDADCF) was determined and found to be 2 moles of DCF produced per mole of hydrogen peroxide used.     

Cell lysis with dimethyl sulphoxide produces stable homogeneous solutions in the dichlorofluorescein oxidative stress assay

The oxidation of 2′,7′-dichlorodihydrofluorescein (2′,7′-dichlorofluorescin, DCFH) to a fluorescent product, 2′,7′-dichlorofluorescein (DCF), is commonly used to quantitatively measure oxidative stress in cells using a fluorescence microplate reader. However, many cell lines tend to grow non-uniformly in the wells. This non-uniform distribution results in a high degree of variability in the fluorescence signal and decreases the precision of the method. Also, samples treated in large culture plates, dishes or flasks cannot be assayed directly in fluorescence microplate readers. This study reports an improved DCF assay method that lyses cells with DMSO/PBS (90% dimethyl sulphoxide/10% phosphate buffered saline). Oxidative stress was induced with either hydrogen peroxide or an hypoxia-reoxygenation treatment. Cell lysis with DMSO/PBS resulted in highly stable fluorescence signals in comparison to Triton X-100/PBS lysed cells. The precision of DCF fluorescence measurements of DMSO/PBS lysed cells was much better than for attached cells measured directly in 96-well plates. While DCF fluorescence in PBS was strongly quenched by albumin, no quenching occurred in DMSO/PBS. In conclusion this study describes a more convenient and accurate method for measuring cellular oxidative stress that also makes it possible to assay cells treated in large culture plates.     

Formation of the superoxide anion radical during the oxidation of biogenic amines catalyzed by mitochondrial monoamine oxidase

The studies on the activity of monoamine oxidase from human placenta, using 2-phenylethylamine as a substrate, corroborate the hypothesis on the possible superoxide radical generation upon FAD oxidation at the second (aerobic) stage of monoamine oxidase reaction. It has been shown that hydrogen peroxide, but not other activated O2 forms, was the end product of this reaction. No superoxide radical generation took place in such systems. And therefore, the induction of lipid peroxidation in the presence of catalase was impossible in mitochondrial membranes containing monoamine oxidase and amines oxidized by it.     

Synthesis and biological applications of two novel fluorescent proteins-labeling probes

Two novel chlorinated fluoresceins 2′,4′,5′,7′-tetrachloro-6-(5-carboxypentyl)-4,7-dichloro fluorescein succinimidyl ester (1G) and 2′,4′,5′,7′-tetrachloro-6-(3-carboxypropyl)-4,7-dichlorofluorescein succinimidyl ester (2G) were synthesized as fluorescent probes for labeling proteins. Structures of target compounds and intermediates were determined via IR, MS, ¹H NMR and element analysis. The investigation in immunofluorescence histochemistry showed them had strong fluorescence, high photostability and good biocompatibility.     

Synthesis of 2′,3′-Didehydro-2′,3′-dideoxyisoinosine and Oxidation of Fluorescent 2-Hydroxypurine Nucleosides by Xanthine Oxidase

Abstract

The syntheses of 2′,3′-didehydro-2′,3′-dideoxyisoinosine (d4isoI, 4) as well as 7-deaza-2′,3′-didehydro-2′,3′-dideoxyisoinosine (d4c₇isoI, 5) are described. Compounds 4 and 5 show both strong fluorescence. Compound 4 is oxidized by xanthine oxidase to give the corresponding xanthine 2′,3′-dideoxy-2′,3′-didehydronucleosides. A preparative chemo-enzymatic synthesis of 2′-deoxyxanthosine (3) is described.     

Assay of biogenic amines by bioluminescence

A bioluminescent procedure to measure noradrenaline and serotonin has been realized. The amines are oxidized by the monoamine oxidase of pig brain mitochondria. The NH3 generated in this reaction is directly measured by enzymatic reaction. The coenzyme of this last reaction is the NADH,H+ which is measured with a bioluminescent system: the FMN-oxidoreductase-luciferase. The extension to other amines is possible, it depends only of the specificity of the monoamine oxidase.     

Potentiometric and colorimetric methods for the assay of 2',3'-cyclic nucleotide 3'-phosphohydrolase

Abstract— A potentiometric titration method for the assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase is presented. Progress curves of the reaction were recorded automatically by pH-stat. 2-Mercaptoethanol was added to the reaction mixture to maintain a linear rate of reaction. The method is suitable for obtaining kinetic parameters and can be used for the rapid assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in nervous tissues. An improved colorimetric method for estimation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity at the optimum pH is described. This method employs the two-step procedure in which decyclization by 2′,3′-cyclic nucleotide 3′-phosphohydrolase and dephosphorylation by Escherichia coli alkaline phosphatase (EC 3.1.3.1) are carried out separately under the optimum conditions for each enzyme. The method is sensitive and most convenient for routine assays.     

Colorimetric assay for monoamine oxidase in tissues using peroxidase and 2,2'-azinodi(3-ethylbenzthiazoline-6-sulfonic acid) as chromogen

Monoamine oxidase is assayed in tissue by a colorimetric reaction using horse radish peroxidase and 2,2'-azinodi(ethylbenzthiazoline-6-sulfonic acid to measure H2O2 formed during oxidation of amines. The method has a coefficient of variation of approximately 2.5% and provides results comparable with those of radiometric assay. Monoamine oxidase activities in rat liver mitochondria and crude mitochondrial fraction from brain and with tyramine as a substrate were 18.9 +/- 0.4 and 4.61 +/- 0.15 nmol/min/mg of protein, respectively, using this method. Kinetic parameters of liver and brain monoamine oxidase with various substrates and inhibitors appeared to be the same when determined by either colorimetric or radiometric methods.     

2′,7′-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy

Abstract

Reactive oxygen species (ROS) are critically important chemical intermediates in biological studies, due to their multiple physiologically essential functions and their often pathologically deleterious effects. Consequently, it is vital that their presence in biological samples has to be quantifiable. However, their high activity, very short life span and extremely low concentrations make ROS measurement a scientifically challenging subject for researchers. One of the widespread methods for ROS detection, based on the oxidation of the non-fluorescent probe 2′,7′-dichlorodihydrofluorescein (DCFH₂) to yield the highly fluorescent 2′,7′-dichlorofluorescein (DCF), was developed more than 40 years ago. However, from its initial application, argumentative questions have arisen regarding its action mechanisms, reaction principles and especially its specificity. Herein, the authors attempt to undertake a comprehensive review: to describe the basic characteristics of DCFH₂; to discuss the present views of the mechanisms of its fluorescence formation; to summarize the fluorescence formation interferents; to outline its application in biological research; and to underline its advantages and disadvantages in ROS detection as well as for the methodological considerations that arise during analysis.     

一种快速非同位素测定2',5'-寡聚腺苷酸合成酶活性的方法

介绍一种新的非同位素测定２′,５′－寡聚腺苷酸合成酶（２′,５′－ＯＡＳＥ）活性的方法．反应液经已糖激酶处理,点样于ＰＥＩ－纤维素薄层层析板上,经甲醇浸泡与预层析和在０．７５ｍｏｌ／ＬＫＨ２ＰＯ４（ｐＨ３．５）缓冲液中的层析可使ＡＤＰ和２′,５′－Ａｎ分离开,系统偏差和２′,５′－ＯＡＳＥ测活分析表明,本方法可用于粗酶液及部分纯化酶液的２′,５′－ＯＡＳＥ活性测定,并可用于临床生化分析     

Pathological changes in platelet histamine oxidases in atopic eczema

Increased plasma histamine levels were associated with significantly lowered diamine and type B monoamine oxidase activities in platelet-rich plasma of atopic eczema (AE) patients. The diamine oxidase has almost normal cofactor levels (pyridoxal phosphate and Cu(2+)) but the cofactor levels for type B monoamine oxidase (flavin adenine dinucleotide and Fe(2+)) are lowered. The biogenic amines putrescine, cadaverine, spermidine, spermine, tyramine and serotonin in the sera, as well as dopamine and epinephrine in EDTA-plasma were found to be normal. It is unlikely, therefore, that these amines are responsible for the decreased activities of monoamine and diamine oxidase in these patients. The most likely causative factors for the inhibition of the diamine oxidase are nicotine, alcohol, food additives and other environmental chemicals, or perhaps a genetic defect of the diamine oxidase.     

Isolation and Characterisation of Two Quercetin Glucosides with Potent Anti-Reactive Oxygen Species (ROS) Activity and an Olean-12-en Triterpene Glucoside from the Fruit of Abelmoschus esculentus (L.) Moench

Abelmoschus esculentus (Okra) is used in the traditional treatment of cancer, hyperlipidaemia and hyperglycaemia. We, therefore, investigated its composition and potential cytotoxic or antioxidant properties that might underlie its phytotherapeutic applications. Its methanolic fruit extract yielded compounds 1 , 2 and 3 , identified through NMR, UV and MS analyses as olean-12-en-3-O-β-d -glucopyranoside, isoquercitrin (quercetin glucoside) and 5,7,3′,4′-tetrahydroxy-flavonol-3-O-[β-d -glucopyranosyl-(1→6)]-β-d -glucopyranoside (quercetin diglucoside), respectively. Following 48 h exposure, oleanene glucoside was mildly toxic to the HeLa and the MRC5-SV2 cancer cells, isoquercitrin was not toxic except at 100 μg/ml in HeLa, and quercetin diglucoside elicited no toxicity. In a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay of intracellular levels of reactive oxygen species (ROS), hydrogen peroxide increased ROS levels, an effect not affected by oleanene glucoside but protected against by isoquercitrin and quercetin diglucoside, with IC₅₀ values, respectively, of 2.7±0.5 μg/ml and 1.9±0.2 μg/ml (3 h post-treatment) and 2.0±0.8 μg/ml and 1.5±0.4 μg/ml (24 h post-treatment.) This is the first report of this oleanene skeleton triterpenoid in the plant. The work provides some insight into why the plant is included in remedies for cancers, cardiovascular complications and diabetes, and reveals it as a potential source of novel therapeutics.     

A rapid,sensitive method for accurate determination of the lecithin/sphingomyelin ratio by thin-layer chromatography and reflectance spectrofluorometry

A highly reproducible thin-layer chromatographic procedure has been developed for accurate determination of the lecithin/sphingomyelin ratio. Two interfering compounds, phosphatidyl inositol and phosphatidyl serine, have been investigated and eliminated by adsorption onto DEAE-cellulose. A uniform fluorescence staining procedure employing 2′,7′-dichlorofluorescein has been developed. Accurate quantitation was performed by direct measurement of the reflected fluorescence intensity of the lecithin and sphingomyelin fluorophore spots with a spectrofluorometer equipped with a thin-layer scanning attachment. Stability and reproducibility studies are reported.     

Role of reactive oxygen species generated by NADPH oxidase in the mechanism of activation of K+-Cl--cotransport by N-ethylmaleimide in HepG2 human hepatoma cells

K⁺-Cl^--cotransport (KCC) is ubiquitously present in all cells, and plays an essential role in ion and volume regulation. In this study we investigated the role of reactive oxygen species (ROS) in regulation of KCC in HepG2 human hepatoblastoma cells. N-ethylmaleimide (NEM), a KCC activator, induced Cl^--dependent K⁺ efflux, which was markedly prevented by KCC inhibitors (calyculin-A, genistein and BaCl₂), indicating that KCC is activated by NEM in the HepG2 cells. Treatment with NEM also induced a sustained increase in the level of intracellular ROS assessed by 2′,7′-dichlorofluorescein flourescence. Antioxidants, N-acetyl cysteine or N,N′-diphenyl-p-phenylenediamine significantly inhibited both ROS generation and KCC activation induced by NEM. The NEM-induced ROS production was significantly suppressed by inhibitors of NADPH oxidase (diphenylene iodonium, apocynin and neopterine). These inhibitors also significantly inhibited the NEM-induced KCC activation. Taken together, these results suggest that ROS generated by NADPH oxidase may mediate the NEM-induced activation of KCC in human hepatoma cells.     

Syntheses,properties, and use of fluorescent N-(5′-phospho-4′-pyridoxyl)amines in assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent K_m value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay.     

Use of bacterial luciferase for determining monoamine oxidase activity

A bioluminescence assay is proposed for measuring monoamine oxidase activity in different biological specimens (platelets, mitochondria). The assay is based on the bioluminescent reaction catalysed by bacterial luciferase and coupled to monoamine oxidase. Two modifications of the bioluminescence assay were used. In the first case, the bioluminescent system was added to monoamine oxidase preincubated with the substrates, while in the second case, all the components of the coupled enzymatic systems were directly mixed in a cell. The proposed bioluminescence assay is simple, highly sensitive and rapid, and could be especially useful for biomedical examinations.     

2-Chloro-2-phenylethylamine as a mechanistic probe and active site-directed inhibitor of monoamine oxidase from bovine liver mitochondria

The reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B was investigated to study the mechanism of this enzyme and its inactivation by this compound. 2-Chloro-2-phenylethylamine is a substrate with a Km of 30 microM and a turnover number of 80 min-1 at pH 6.5 at 30 degrees C. Incubation of 2-chloro-2-phenylethylamine with the enzyme led to the normal oxidation product, 2-chloro-2-phenylacetaldehyde, but only traces (0.25 mol%) of 2-phenylacetaldehyde, the product anticipated if the oxidation of substrate involved a stabilized carbanion at C-1 and elimination of chloride ion. These data suggest that a carbanion is not a likely intermediate in the oxidation of amines by monoamine oxidase. During the mechanistic studies we noted time-dependent inactivation of monoamine oxidase B by 2-chloro-2-phenylethylamine under both aerobic and anaerobic conditions. Inactivation was not reversible. Aerobically 2-chloro-2-phenylethylamine is oxidized to 2-chloro-2-phenylacetaldehyde which covalently modifies the enzyme (tau 1/2 = 40 min). Benzyl alcohol, a substrate analog, gives substantial protection against inactivation under aerobic conditions (tau 1/2 = 320 min), suggesting that an active site residue is modified. Anaerobic reaction of 2-chloro-2-phenylethylamine with monoamine oxidase B probably proceeds by direct alkylation of an enzyme residue (tau 1/2 = 140 min). Reduction with [3H]NaBH4 of the inactivated enzyme gave from 0 to 0.7 and from 4.5 to 5.6 mol of hydride incorporation for enzyme inactivated anaerobically and aerobically, respectively. The latter results are in agreement with inactivation by unmodified inhibitor and inactivation by oxidized inhibitor for the anaerobic and aerobic reactions, respectively. It is suggested that 2-chloro-2-phenylethylamine or its oxidation product 2-chloro-2-phenylacetaldehyde may serve as an active site affinity reagent for monoamine oxidase.     

Photoreduction of the fluorescent dye 2′-7′-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

The photoreduction of 2′-7′-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/^·OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.     

A new spectrophotometric assay for 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in nervous tissue

A new continuous spectrophotometric method for determining 2′,3′-cyclic nucleotide 3′-phosphohydrolase (EC 3.1.4.37) is described. The assay method involves monitoring the decrease in pH which accompanies the hydrolysis of 2′,3′-cyclic AMP. The reaction is performed in the presence of phenol red and the pH change is followed spectrophotometrically by recording the decrease in absorbance of the basic chromophore at 560 nm. The assay method is sufficiently sensitive to make accurate determinations of CNPase activity in 20-μl samples of CNS homogenates containing less than 5 μg protein. The primary advantage of the phenol red CNPase assay is the ease and speed with which it is performed.     

5′-Phosphonates of Ribonucleosides and 2′-Deoxyribonucleosides: Synthesis and Antiviral Activity

Abstract

5′-Phosphonates of natural 2′-deoxynucleosides and ribonucleosides were synthesized by condensation of 3′-O-acylated 2′-deoxynucleosides or 2′,3′-substituted (2′,3′-O-isopropylidene, 2′,3′-O-methoxymethylene or 2′,3′-O-ethoxymethylene) ribonucleosides. As condensing agents, either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulphonyl chloride were used. Nucleoside 5′-ethoxycarbonylphosphonates were converted into corresponding nucleoside 5′-aminocarbonylphosphonates by action of ammonia in methanol or aqueous ammonia. 5′-Hydrogenphosphonothioates of thymidine and 3′-deoxythymidine were obtained by reaction of phosphinic acid in the presence of pivaloyl chloride with 3′-O-acetylthymidine or 3′-deoxythymidine, respectively, followed by addition of powedered sulfur. 5′-O-methylenephosphonates of thymidine and 2′-deoxyadenosine were prepared by intramolecular reaction of corresponding 3′-O-iodomethylphosphonates under basic conditions. All compounds were tested for inhibition of several viruses, including HSV-2 and CMV, but showed no activity. A few compounds insignificantly inhibited HIV-1 reproduction. Thymidine 5′-hydrogenphosphonate neutralized anti-HIV action of 3′-azido-3′-deoxythymidine (AZT) and it indirectly showed that even some nucleoside 5′-phosphonates could be partly hydrolyzed in cell culture to corresponding nucleosides.

5′-Phosphonates of modified 2′-deoxynucleosides in which one group in a phosphate residue is substituted for hydrogen, alkyl or other groups, have shown to be potent biologically