title: Coupling of size-exclusion chromatography to a continuous assay for subtilisin using a fluorescence resonance energy transfer peptide substrate: testing of two standard inhibitors.
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 approximately 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 microM in the flow injection analysis (FIA) mode and 12 microM in the on-line LC mode. System validation was performed by determining IC50 values for aprotinin for the FIA mode (19 microM) and the on-line mode (22 microM). These IC50 values were in line with the value determined in batch experiments (25 microM). With this system, chemical information (i.e., chromatographic retention time) and biological information (i.e., enzyme inhibition) can be combined to characterize mixtures.
authors.affiliation: Department of Analytical Chemistry and Applied Spectroscopy, Division of Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. hirata@few.vu.nl
authors.forename: Junko
authors.initials: J
authors.lastname: Hirata
authors.forename: Lai Ping
authors.initials: LP
authors.lastname: Chung
authors.forename: Freek
authors.initials: F
authors.lastname: Ariese
authors.forename: Hubertus
authors.initials: H
authors.lastname: Irth
authors.forename: Cees
authors.initials: C
authors.lastname: Gooijer
mesh_terms: D007611:Aprotinin / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D020128:Inhibitory Concentration 50; D009842:Oligopeptides / Q000378:metabolism*; D009862:Online Systems; D013381:Subtilisins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D013450:Sulfones / Q000494:pharmacology
chemical_list: C505205:HIV protease substrate 1; D009842:Oligopeptides; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D007611:Aprotinin; D013381:Subtilisins