Abstract

RAMIREZ ARISTIZABAL, Luz Stella; ARIAS, Cristian Mauricio  and  MARULANDA OSORIO, Jackeline. Standarization of a spectrofluorometric method to measure secreted aspartic protease (Sap) from Candida albicans. Rev Cubana Farm [online]. 2014, vol.48, n.2. ISSN 0034-7515.

Introduction: the Secreted Aspartyl Protease (Sap) is considered a virulence factor in the infection process caused by Candida albicans. The frequency of candidiasis worldwide is increasing, hence the need for finding new drugs to fight this illness and also a quick and economic evaluation and screening methods to identify some Sap inhibition agents. Objective: to standardize fluorescent method for identifying the Sap activity inhibition. Methods: Sap production was induced in C. albicans cultures following the methodology described by Capobianco, their levels were evaluated by electrophoresis on SDS-PAGE gels at different periods of time. Sap activity was checked by spectrofluorometry, for which the reaction conditions were determined, varying the concentrations of copper and fluorexon, and the inhibition results were expressed as decreased fluorescence signal. Pepstatin A served as Sap inhibition control whereas pancreatin was the positive control of the protease activity. Results: the concentration rates of 5.5 µM and 5.0 µM were found for fluorexon and copper, respectively; their optimal coupling times was 120 min and the maximum Sap activity was observed after 22 h of incubation. Conclusions: the standardized conditions for the spectrofluorometric method allowed confirming the inhibition of Sap by Pepstatin A and showing that this is a viable method to evaluate inhibitors of this protease.

Keywords : Candida albicans; inhibition; spectrofluorometric method; secreted aspartyl protease.

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