Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10548042 | Journal of Chromatography A | 2005 | 5 Pages |
Abstract
Liquid chromatography (LC) was coupled on-line to a homogeneous continuous-flow protease assay using fluorescence resonance energy transfer (FRET) as a readout for the screening of inhibitors of an enzyme (e.g., Subtilisin Carlsberg). The inhibitors aprotinin (a protein of â¼6500 g/mol) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 240 g/mol) were mixed with other, non-active compounds and separated on a size-exclusion chromatography column. After the separation, the analytes were eluted to the postcolumn reactor unit where the enzyme solution and subsequently the FRET peptide substrate were added; by measuring the fluorescence intensity the degree of inhibition was monitored on-line. As expected, only the two inhibitors caused a change in the FRET response. Detection limits for aprotinin were 5.8 μM in the flow injection analysis (FIA) mode and 12 μM in the on-line LC mode. System validation was performed by determining IC50 values for aprotinin for the FIA mode (19 μM) and the on-line mode (22 μM). These IC50 values were in line with the value determined in batch experiments (25 μM). With this system, chemical information (i.e., chromatographic retention time) and biological information (i.e., enzyme inhibition) can be combined to characterize mixtures.
Related Topics
Physical Sciences and Engineering
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Analytical Chemistry
Authors
Junko Hirata, Lai Ping Chung, Freek Ariese, Hubertus Irth, Cees Gooijer,