Continuous-flow protease assay based on fluorescence resonance energy transfer |
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Authors: | Junko Hirata Freek Ariese Cees Gooijer Hubertus Irth |
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Institution: | Department of Analytical Chemistry and Applied Spectroscopy, Division of Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands |
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Abstract: | A homogeneous continuous-flow assay using fluorescence resonance energy transfer (FRET) for detection was developed to measure the hydrolysis of HIV Protease Substrate 1 (to which two choromophores, EDANS and DABCYL are covalently attached) by a protease (e.g. Subtilisin Carlsberg) and the influence of inhibitors. In the continuous-flow assay, an inhibitor solution and an enzyme solution were first eluted into the system and allowed to react with each other in a reaction coil. Subsequently, the substrate solution was added to an enzyme-inhibitor mixture in a second reaction coil and incubated for 1 min. Finally, the fluorescence intensity was monitored.The system was also utilized to measure the inhibition of the protease by two weak acidity inhibitors which are 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and ethylenediaminetetraacetic acid (EDTA). Using the obtained optimum conditions for AEBSF, a detection limit of 0.3 mmol/l was achieved and the relative standard deviation was below 3.7% in the 2.5-7.5 mmol/l range. For EDTA, which required a 20 times higher substrate concentration than AEBSF, a detection limit of 0.2 mmol/l was obtained and the relative standard deviation was below 9.6% in the 0.5-7.5 mmol/l range.The optimization of pH, substrate concentration, enzyme concentration, reaction time and temperature are described. Organic modifier effects were also investigated. Methanol, acetonitrile and DMSO could be tolerated up to 30%. |
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Keywords: | Flow injection analysis FIA FRET HIV Protease Substrate 1 Continuous flow |
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