A sensitive method for the assay of monoamine oxidases A and B is described which employs high-performance liquid chromatography with fluorescence detection. Rat brain mitochondria were used as a preparation of the enzymes. p-Sulfamoylbenzaldehyde and benzaldehyde formed enzymatically from p-sulfamoylbenzylamine (the substrate of monoamine oxidase A) and benzylamine (the substrate of monoamine oxidase B), respectively, are converted simultaneously into fluorescent compounds with 2,2'-dithiobis(1-aminonaphthalene). These compounds are separated by reversed-phase chromatography on mu Bondapak CN. The limits of detection for p-sulfamoylbenzaldehyde and benzaldehyde formed enzymatically are 30 and 10 pmol per assay tube, respectively. 相似文献
A simple ultraviolet spectrophotometric method for the assay of microgram amounts (0.1–25 μg/ml) of 16 different p-aminobenzenesulfonamides (sulfonamides) in 0.1 N sodium hydroxide has been developed. The method is simple, accurate, and highly sensitive within the limits of the experimental conditions described and can be used in pharmacies for the assay of sulfonamides in tablets and in pharmaceutical industries for quality control or environmental monitoring as well. The present method is more rapid than any analytical assay reported for these compounds thus far. The values of λmax and ?max for these sulfonamides have also been presented. 相似文献
Immobilized bovine plasma and pig kidney amine oxidases coupled with the hydrogen peroxide-peroxidase system are proposed for the detection of di- and polyamines in biological samples. The measurements were carried out spectrophotometrically with a flow reactor filled with the enzymes immobilized on Sepharose. The simplicity of the procedure and the short analysis time are the main advantages. The kinetic characteristics of the immobilized system, amine oxidases and peroxidase, are also reported. This method, which appears useful for routine analyses, permits the determination of polyamines in tissue homogenates and isolated cells. 相似文献
This study describes the design of sensitive, selective, and fluorogenic reporter substrates for monoamine oxidase (MAO) enzymes. This was achieved by an iterative effort, guided by PET and TICT photophysical concepts, which led to the development of irreversible redox switches based on a facile oxidation-cyclization reporting mechanism. Specifically, enzymatic oxidation of the ethylamino group in probe 9 proceeded via a putative aldehyde intermediate, which subsequently underwent spontaneous and intramolecular condensation with the aniline amino group furnishing an indole product in an irreversible fashion. This overall change resulted in a significant change in the emission intensity. When expressed in terms of brightness, the origins of this emission switch may be rationalized by the changes in quantum yield and absorbance strength. The fluorescence readout directly correlated with the kinetics of the oxidative step (i.e., reporting mechanism was fast, the intermediate aldehyde was not detected). Probe 9 is a good substrate for MAO B (Km = 510 +/- 40 muM, kcat = 21 min-1) with the kinetic parameters comparable to physiological substrates. This probe not only allows for direct and continuous measurement of MAO activity in mitochondria and tissue homogenates, but more importantly sets the stage for future studies in intact cells and organs. 相似文献
A sensitive phospholipase A assay suitable for organelle activities is described. Activation of the enzyme produces an increase in membrane lysophospholipids. The amino groups of extracted lipids were derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and the amounts of phosphatidylethanolamine and its lysoform were quantitated using HPLC equipped with a SiO2 column and a fluorescence detector. The procedure was tested with porcine enzyme acting on liposomes and mitochondria, and with endogenous mitochondrial enzyme. 相似文献
Summary Three reagents and a variety of fluorimetric methods are introduced for the assay of-glycolic compounds, polar olefinic compounds, and olefins. The procedures are based on the controlled oxidation of these compounds to aldehydes and analysis of the aldehydes with J-acid, 2,4-pentanedione, or dimedone. Most of the methods show reasonable sensitivity and accuracy and should be capable of use in air pollution studies. Recommendations are made for their use. Results with these methods confirm the presence of large amounts of-glycolic compounds in aqueous extracts of urban airborne particulates.
Zusammenfassung Drei Reagenzien und verschiedene fluorimetrische Methoden zur Analyse von-Glykolen, polaren ungesättigten Verbindungen und Olefinen wurden vorgeschlagen. Diese Verfahren beruhen auf der kontrollierten Oxydation der genannten Verbindungen zu Aldehyden und deren Analyse mit J-Säure (Aminonaphtholsulfonsäure), 2,4-Pentandion oder Dimedon. Meistens sind diese Methoden sehr empfindlich und genau; sie dürften sich für das Studium der Luftverunreinigung eignen. Angaben hierfür werden gemacht. Die Ergebnisse bestätigen die Anwesenheit großer Mengen von-Glykolverbindungen in wäßrigen Stadtluftextrakten.
AbstractIn this study, a novel series of benzothiazole-thiazolylhydrazine (3a–3i) was synthesized and their structures were characterized by 1H-NMR, 13C-NMR spectrometry, and mass spectroscopy. These compounds were evaluated as inhibitors of type A and type B monoamine oxidase (MAO) enzymes. The most active compound 3b (2-((2-(2-(4-(4-Nitrophenyl)thiazol-2-yl)hydrazineylidene)-2-phenylethyl)thio)benzothiazole) showed strong inhibitory activity at hMAO-A (IC50 of 0.095?±?0.004?µM). Furthermore, compound 3i (2-((2-(2-(4-(2,4-dichlorophenyl)thiazol-2-yl)hydrazineylidene)-2-phenylethyl)thio)benzothiazole) showed significant inhibition profile on hMAO-A with the IC50 values 0.141?±?0.006?µM. 相似文献
Extended cavity calix[4]pyrroles and a calix[6]pyrrole were synthesized by cyclization of 5-methyl-5-(4-nitrophenyl)dipyrromethane with acetone in the presence of acid. The solid-state structures of the novel macrocycles were determined by X-ray crystallography. The host-guest chemistry of these receptors towards halide ions was investigated in solution by 1H NMR titration techniques and compared with those of the meso-octamethylcalix[4]pyrrole and meso-dodecamethylcalix[6]pyrrole. The binding of chloride anions was observed to occur with different affinities on the two faces of the novel calix[6]pyrrole derivative described here. 相似文献
Fluorescamine was subjected to reaction with dopamine and norepinephrine (catecholamines) and with 3-methoxytyramine and normetanephrine (3-methyl metabolites of catecholamines) in phosphate or borate buffer. Catecholamines gave the highest fluorescent intensity at pH 8.0 in phosphate buffer but lower fluorescence in borate buffer. The fluorophores produced in phosphate or borate buffer were the same but the fluorescence intensities were suppressed in borate buffer. The dopamine and norepinephrine fluorophores were separated by high-pressure liquid chromatography on Hitachi 3011 gel with methanol-0.10 M Tris buffer of pH 8.0 (7:3). They were measurable at the 100-pmole level. The metabolites were also measurable by the same chromatography. By using methanol-0.15 M borate buffer of pH 8.0, cate-chol-O-methyltransferase activity might be assayed. 相似文献
High profile: new activity-based protein profiling (ABPP) probes have been designed that target exclusively monoamine oxidases A and B within living cells (see picture; FAD=flavin adenine dinucleotide, FMN=flavin monodinucleotide). With these probes it could be shown that the MAO inhibitor deprenyl, which is in clinical use against Parkinson's disease, shows unique protein specificity despite its covalent mechanism of action. 相似文献
A fluorimetric method for the assay of argininosuccinate lyase in human plasma (15–150 units) and erythrocytes (40–1200 units) is established. The arginine formed enzymatically is quantified by means of its fluorescent reaction with benzoin. The method is rapid, simple and sensitive enough to allow the enzyme activity to be determined in as little as 100 μl of plasma or 12.5 μl of packed erythrocytes. 相似文献
This work reports a hybrid flow-injection analysis (FIA)/sequential-injection analysis (SIA) method for the rapid enzymatic assay of glucose with soluble glucose oxidase (GOD). The method relies on the sequential injection of segments of the sample and of a solution of enzyme by means of a multi-port selection valve in a flowing water stream. As the two zones are swept downstream, they overlap and merge so that the glucose in the sample is enzymatically oxidised. The generated hydrogen peroxide is merged with an alkaline luminol solution and the chemiluminescence (CL) intensity is monitored and related to the glucose concentration in the sample. The linear range of the method for glucose determination is 0.01-1 mmol L−1, the relative standard deviation is 3.9% at the 0.08 mmol L−1 level (n = 8), the limit of detection at the 2σ level is 4 μmol L−1 glucose and the injection rate is 80 h−1. The method was applied to the analysis of energy drinks and honey with relative errors in glucose determination in the range 100 ± 4.3%. The advantages of the proposed method are the wide linear range, the simple instrumentation used, the low consumption of sample and reagents, the elimination of catalysts and immobilised enzymes and the high sample throughput. 相似文献
A new solid-phase enzyme-linked competitive binding assay for vitamin B12 (cyanocobalamin) is described. The assay is based on the competition between analyte B12 molecules and a glucose-6-phosphate dehydrogenase-vitamin B12 conjugate for a limited number of R-protein binding sites immobilized on sepharose particles. After appropriate incubation and washing steps, the enzyme activity bound to the solid-phase is inversely related to the concentration of B12 in the sample. Under optimized conditions, the method can detect B12 in the range of 3×10–10–1×10–8M (using 100l sample) with high selectivity over other biological molecules. 相似文献
The mono/bis ratio for the Suzuki-Miyaura cross coupling of p-dibromobiphenyl and p-dibromo(penta-p-phenylenes) with arylboronic acids and esters has been studied. The coupling reaction is demonstrated to be highly selective for monoarylation when the substrate is a p-dibromooligoarene, while selective biarylation is obtained for p-diiodoterphenyl. The mono/bis coupling-ratio for these compounds was highly sensitive to the nature of the halogen involved, however steric hindrance or electronic characteristics of the boronic derivative did not affect the selectivity of the reaction. The reaction yields observed were higher at room temperature and when arylboronic pinacol esters were used. These reactions also offer a useful method for the preparation of asymmetrically substituted terphenyls and hexa-p-phenylenes, giving good yields. 相似文献
Nitrogen kinetic isotope effects for the oxidation of benzylamine and (1,1-(2)H(2))benzylamine by recombinant human monoamine oxidase B show that cleavage of the CH bond is not concerted with rehybridization of the nitrogen atom. 相似文献