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  • 1
    Electronic Resource
    Electronic Resource
    Phenolic substrates for fluorometric detection of laccase activity (1997)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 153 (1997), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: A fluorometric procedure has been developed for detection and estimation of laccase activity in fungal broth cultures. Laccase solution was pretreated with catalase for 1 h at 37°C and pH 5. Homovanillic acid was then added and the reaction mixture incubated for a further hour at 37°C. The fluorescence was then developed by addition of 0.1 M glycine buffer at pH 10. Laccase preparations from Pyricularia oryzae, Coriolus hirsutus and Pycnoporus cinnabarinus catalysed formation of a fluorescent product of HVA but the optimum pH values of enzyme activities varied. The culture fluids of several other fungi also catalysed development of fluorescence in solutions containing HVA. p-Hydroxyphenylacetic acid was a poor substrate for all laccases in vivo except that produced by Perennipora tephropora.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1997.tb12614.x
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  • 2
    Electronic Resource
    Electronic Resource
    Analysis of reduced glutathione using a reaction with 2,4′-dichloro-1-(naphthyl-4-ethoxy)-s-triazine (EDTN) (1999)
    Springer
    Microchimica acta 130 (1999), S. 225-231 
    Details
    ISSN: 1436-5073
    Keywords: 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine ; EDTN ; reduced glutathione ; EDTN-glutathione analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A fluorescent adduct was formed between 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine (EDTN) and reduced glutathione in a reaction at 37 °C and pH 9.2. This reaction was used as the basis of an assay for reduced glutathione. The fluorescence was examined at an excitation wavelength of 319 nm and an emission wavelength of 425 nm after extraction of residual unreacted EDTN with methylene dichloride and subsequent dilution of the aqueous phase with ethanol containing 0.01 percent Triton X-100. The reaction rate was low at pH 7 but was accelerated by addition of preparations containing the enzyme glutathione-S-transferase. The adduct gave a discrete peak using isocratic elution with HPLC on a Nova-pak C18 3 μm reverse phase column and a solvent system of methanol: 0.1 M phosphate buffer pH 6.3 (40∶60). An analytical concentration range of 24 to 240 μM reduced glutathione was obtained with an ultraviolet detection system but the concentration range was 7.5 to 75 μM when a fluorescence detection system was used. Adducts of other mercapturic acid pathway thiol compounds were not formed at 37 °C under the conditions used and hence did not interfere in the assay. They were formed by heating EDTN and the respective thiol compound at 60 °C for 30 min and they clearly separated from the reduced glutathione compound on HPLC analysis. A strong reaction was observed with digitonin while solutions of tyrosine, at 10 mM concentration, also reacted but these reactants are unlikely to interfere with reduced glutathione analysis in biological systems. When adduct formation was used to estimate reduced glutathione concentrations in some mammalian and plant tissues the reaction using 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine and HPLC separation gave the same results as ano-phthaldialdehyde assay for liver and muscle but the HPLC method gave slightly lower values for other mammalian and plant tissues. The differences were attributed to other material in the tissue extracts which was fluorescing at the same wavelengths as the reduced glutathione adduct.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01242910
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  • 3
    Electronic Resource
    Electronic Resource
    Estimation of hydrogen peroxide formed and residual ascorbate in the copper catalysed oxidation reaction of ascorbate at pH 7 (1992)
    Springer
    Microchimica acta 106 (1992), S. 143-152 
    Details
    ISSN: 1436-5073
    Keywords: ascorbate ; hydrogen peroxide ; copper ; fluorescent assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A fluorescent method has been developed for determination of ascorbic acid concentrations. The method involves treatment of the ascorbic acid with Cu(II) and treating the hydrogen peroxide formed with horseradish peroxidase in the presence ofp-hydroxyphenylacetic acid, to form a fluorescentp-hydroxyphenylacetic acid dimer. The reaction is suitable for analysis of concentrations in the range from 50 μM to 4 mM ascorbic acid solutions and can be used for analysis of pharmaceutical preparations but is unsuitable for analysis of ascorbate in preparations derived from natural sources. By using a slight modification of the analytical technique it is possible to measure the amount of hydrogen peroxide formed and the residual concentrations of ascorbic acid in solutions treated with varying amounts of copper ion.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01242085
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  • 4
    Electronic Resource
    Electronic Resource
    Isolation of the 9-fluorenylmethyl chloroformate derivative in the determination of sulphamethazine (1998)
    Springer
    Microchimica acta 128 (1998), S. 31-36 
    Details
    ISSN: 1436-5073
    Keywords: sulphamethazine ; FMOC ; RPLC ; amino acids ; sulphonamides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Derivatisation of amine-containing analytes with 9-fluorenylmethyl chloroformate (FMOC) to form fluorescent adducts requires a large excess of FMOC. This excess hydrolyses to form FMOC-OH, which is also fluorescent. Solvent extraction has been investigated as a means of isolating the sulphamethazine (SMZ) adduct (FMOC-SMZ) from the hydrolysis product in order to perform rapid spectrophotometric or spectrofluorimetric assays. However, even under the most favourable pH conditions possible, FMOC-OH was not totally removed. Attempts to enhance the separation by reaction of FMOC-OH with 1-ethoxy-4-dichloro-S-triazinylnaphthalene (EDTN) or by acetylation were also unsuccessful. On the other hand, reaction of FMOC with mixed substrates, followed by two pentane extractions to remove the excess FMOC and direct injection into an HPLC provides the desired separations on a reversed phase column (RPLC) with methanol-modified, (pH 3.5) phosphate buffers. FMOC-SMZ is readily separated from FMOC-OH under all elution conditions, from the FMOC-amino acids (under gradient conditions or isocratically up to 75% methanol), and from other FMOC-sulphonamides and FMOC-dihydrofolate reductase inhibitors (isocratically up to 70% methanol). Hence conversion to the FMOC derivatives permits SMZ to be separated from all of the potential interferants tested by isocratic elution with 70% methanol in RPLC. Analysis for the amino acid derivatives of FMOC may be done without interference from SMZ in samples.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01242186
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