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

• Published in: Revista do Instituto de Medicina Tropical de São Paulo
• Main Authors: Cheila Denise Ottonelli Stopiglia, Mariana Carissimi, Tatiane Caroline Daboit, Valter Stefani, Valeriano Antonio Corbellini, Maria Lucia Scroferneker
• Format: Article in Journal/Newspaper
• Language: English
• Published: Universidade de São Paulo (USP) 2013
• Subjects: Sporotrichosis; Enzymatic activity; Fluorogenic substrate; 6-nitrocoumarin; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1590/S0036-46652013000500010; https://doaj.org/article/828048c06ac04283a47869ec2ce8a061

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 °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.