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11.
Samuel K Yin W Stearns RA Tang YS Chaudhary AG Jewell JP Lanza T Lin LS Hagmann WK Evans DC Kumar S 《Journal of mass spectrometry : JMS》2003,38(2):211-221
Metabolic activation of drug candidates to electrophilic reactive metabolites that can covalently modify cellular macromolecules may result in acute and/or idiosyncratic immune system-mediated toxicities in humans. This presents a significant potential liability for the future development of these compounds as safe therapeutic agents. We present here an example of an approach where sites of metabolic activation within a new drug candidate series were rapidly identified using online liquid chromatography/multi-stage mass spectrometry on an ion trap mass spectrometer. This was accomplished by trapping the reactive intermediates formed upon incubation of compounds with rat and human liver microsomes as their corresponding glutathione conjugates and mass spectral characterization of these thiol adducts. Based on the structures of the GSH adducts identified, potential sites and mechanisms of bioactivation within the chemical structure were proposed. These metabolism studies were interfaced with iterative structural modifications of the chemical series in order to block these bioactivation sites within the molecule. This strategy led to a significant reduction in the propensity of the compounds to undergo metabolic activation as evidenced by reductions in the irreversible binding of radioactivity to liver microsomal material upon incubation of tritium-labeled compounds with this in vitro system. With the efficiency and throughput achievable with such an approach, it appears feasible to identify and address the metabolic activation potential of new drug leads during routine metabolite identification studies in an early drug discovery setting. 相似文献
12.
An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalysed oxidation of homovanillic acid is utilized to determine the concentration of H2O2. The membrane-bound enzyme is attached to a bifurcated fibre bundle permitting excitation and detection of the fluorescence by a fluorometer. The response of the sensor is linear from 1 to 130 M hydrogen peroxide; the coefficient of variation is 3%. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 M and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum. 相似文献
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Enzymatic determination of phenols using peanut peroxidase 总被引:4,自引:0,他引:4
The influence of phenol and its derivatives on the kinetics of oxidation of aryldiamines (indicator-substrates) catalyzed by novel plant peroxidase—cationic peanut peroxidase—was studied. The character of influence of phenols on the kinetics of enzymatic oxidation of benzidine, o-dianisidine, and 3,3′,5,5′-tetramethylbenzidine (TMB) with hydrogen peroxide was found to depend on a correlation between redox properties of phenols and the indicator-substrate of peroxidase. Thus, the catalytic activity of peanut peroxidase is inhibited by phenols with redox potentials higher than that of aryldiamines mentioned above, whereas phenols with potentials below those of aryldiamines, play the role of second substrates of the enzyme. The enzymatic procedures for the determination of numerous phenols on the level of their concentrations 0.05–80 μM were developed using the reactions of benzidine, o-dianisidine, and TMB oxidation. Different analytical signals—the indicator reaction rate and the induction period duration—were used for the determination of phenols, belonging to various groups—the inhibitors and second substrates of the enzyme, respectively. 相似文献
15.
铬(Ⅵ)与谷胱甘肽作用及其中间态配合物形成的电化学表征 总被引:2,自引:0,他引:2
本文采用电化学方法,对谷胱甘肽(GSH)与重铬酸钾的相互作用及其中间态配合物的形成过程进行研究。结果表明:在pH=5.6的HAc-NaAc缓冲溶液中,GSH浓度为Cr(Ⅵ)浓度5倍以上时,Cr(Ⅵ)与GSH作用完全并形成一新的中间态配合物,该中间态配合物于+0.21 V和+0.36 V(vs SCE)处产生一对新的氧化还原峰,UV-Vis的吸收光谱进一步证明了中间态配合物的形成。该配合物不稳定,在一定时间内缓慢分解,其电化学与UV-Vis光谱动力学信息同步。进一步探讨了GSH与Cr(Ⅵ)作用的电极反应机理。当Zn(Ⅱ)存在于该体系时,Zn(Ⅱ)对中间态配合物的生成和分解过程起着双向催化作用。 相似文献
16.
Francesco Trotta Giancarlo Cravotto Stefano Rossignoli 《Journal of inclusion phenomena and macrocyclic chemistry》2002,44(1-4):293-296
Oxidative couplings of 2-naphthol, 6-bromo-2-naphthol and2-naphthylamine were achieved at room temperature in the presence of H2O2, horseradish peroxidase and a suitable cyclodextrin.2-Thionaphthol behaved differently, yielding the corresponding disulfide. Yields of binaphthyl derivatives were generally excellent, and a fairly good enantiomeric excess was observed. Under similar reaction conditions methyl 2-(6-methoxy-2-naphthyl) propanoate, when treated with esterase in the presence of cyclodextrin, yielded naproxen (a well-known anti-inflammatory drug) with a good enantiomeric excess. No reaction product was detected in the absence of cyclodextrin. Cyclodextrins do not act as simple transfer agents. 相似文献
17.
V. Vojinovi? 《Analytica chimica acta》2006,565(2):240-249
A new alternative method for bioprocess monitoring based on bienzymatic analytical microreactors integrated in a flow injection analysis (FIA) system is described. Glucose-, alcohol-, lactate-, galactose- and l-amino acid oxidases (GO, AO, LacO, GalO and LAAO) and horseradish peroxidase (HRP) are immobilized on controlled pore glass (CPG) and used for the development of glucose, ethanol, lactate, galactose and amino acid sensors. The analytical methodology is based on HRP catalysed reaction of hydrogen peroxide produced by oxidases with phenol-4-sulfonic acid and 4-aminoantipyrine. The immobilized enzymes are characterized and used for preparation of the packed bed analytical microreactors. Shelf life and operational stability of the microeactors are determined. GO/HRP, AO/HRP and LAAO/HRP microreactors showed excellent shelf life, they could be stored and reused for more than 6 months with no or very little activity loss, while GalO/HRP and LacO/HRP could be stored for shorter periods of time (10-20 days). Operational stability of GO and LacO microreactors was very good: an equivalent to 16,900 FIA injections of 25 μl to a LacO microreactor resulted in loss of half of its activity, immobilized GO was so stable that it was impossible to evaluate enzyme halflife. Immobilized GalO and LAAO lose their operational activity much faster: approximately 1400 and 8000 FIA injections of the respective substrate solution in a FIA set-up resulted in 50% activity loss. The methods with all the described microreactors were successfully validated using off-line samples from S. cerevisiae, E. coli and mesenchymal stem cell cultures with HPLC as the reference method. 相似文献
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19.
Yannis Dotsikas 《Analytica chimica acta》2004,509(1):103-109
This study describes the employment of a novel imidazole-substituted phenol [4-(1-imidazolyl)phenol] as a highly potent signal enhancer in a horseradish peroxidase (HRP)-luminol chemiluminescence (CL) immunoassay. This competitive-type immunoassay for the model antigen fentanyl is based on the use of fentanyl polyclonal antibody immobilized on white microtiter plates and a biotinylated bovine serum albumin (BSA)-fentanyl derivative as a tracer. The latter was detected by means of streptavidin labeled with HRP, resulting in the generation of a high-intensity and relatively stable chemiluminescent signal, immediately after the addition of the substrate solution (NOAS). The developed method fulfilled the requirements of accuracy (percentage recovery ranged from 93.8 to 107%) and precision (intra- and inter-assay CVs were 2.5-5.2 and 4.5-11.9%, respectively). Its plasma detection limit (1.05 pg ml−1) was lower than those of previous immunoassays. The novel assay was compared in terms of sensitivity and concentration range with other common HRP substrate systems: luminol-p-iodophenol-H2O2 and TMB-H2O2. Finally, the described method was compared with an HRP-fentanyl conjugate-based assay, similar to commercially available kits (SKIT), employing the novel substrate solution for both assays and the differences observed were explained by applying previously described models. The detection limit was 4.82 pg ml−1 for SKIT, recovery values were 94.2-105% and intra- and inter-assay CVs were 2.5-5.2 and 4.5-11.9%, respectively. In conclusion, the proposed assay could be utilized for a wide range of molecules and replace the existing enzyme-labeled antigen-based kits. 相似文献
20.
化学发光单孵多层免疫技术检测粪样中轮状病毒 总被引:3,自引:0,他引:3
以酶联免疫吸附分析(ELISA)夹心测定粪样中轮状病毒(RV)实验为模型,将传统又孵式免疫操作变成单孵式,即在包被RV抗体Ab的微孔中同时加入含RV的粪样上清液和根过氧化物酶(HRP)标记抗RV抗原共同孵育,其结果在载体表面形成多层酶免疫复合物,以HRP催化鲁米诺-对碘苯酚-过氧化氢化学体系作为最终信号检测系统,从而建立起化学发光单孵多层免疫技术(CL-SIMIT)。其灵敏度是ELISA的16倍, 相似文献