Coupling of size-exclusion chromatography to a continuous assay for subtilisin using a fluorescence resonance energy transfer peptide substrate: testing of two standard inhibitors

J. Hirata, L.P. Chung, F. Ariese, H. Irth, C. Gooijer

Research output: Contribution to JournalArticleAcademicpeer-review

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 ∼6500 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 IC
Original languageEnglish
Pages (from-to)140-4
JournalJournal of Chromatography A
Volume1081
Issue number2
DOIs
Publication statusPublished - 2005

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