APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

作者：Ottonelli Stopiglia Cheila Denise; Carissimi Mariana; Daboit Tatiane Caroline; Stefani Valter; Corbellini Valeriano Antonio; Scroferneker Maria Lucia^*

来源：Revista do Instituto de Medicina Tropical de Sao Paulo, 2013, 55(5): 353-356.

DOI：10.1590/S0036-46652013000500010

• Sporotrichosis
• Enzymatic activity
• Fluorogenic substrate
• 6-nitrocoumarin

Introduction: Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods: Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 degrees C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane: ethyl acetate 1: 4 unveiled with UV light. Results: All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion: The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

• 出版日期2013-10