ABSTRACT
BACKGROUND: Fluorochrome-labeled inhibitors of caspases (FLICA) have been designed as an alternative tool for the detection of caspase activation in whole cells. They should label the active site of the corresponding caspase through a covalent attachment to the reactive cysteine residue. METHODS: One of the FLICAs, FAM-DEVD-fmk, was used to monitor apoptosis progression in leukemic JURL-MK1 cells by means of flow cytometry. The effects of unlabeled caspase inhibitors z-DEVD-fmk and z-VAD-fmk on FLICA staining were analyzed to evaluate the contribution of caspase-bound FLICA to the fluorescent signal. Covalent binding of inhibitors to caspase-3 subunit was revealed by Western blotting. RESULTS: Although the unlabeled inhibitors irreversibly bind to caspase-3, completely inhibit its activity, and prevent FLICA binding to caspase-3 even at concentrations lower than 5 muM, they have no effect on FLICA staining of apoptotic cells. CONCLUSIONS: Fluorescent signal of FLICA is characteristic for apoptotic cells but originates mainly from yet unspecified site(s) that differ from the caspase active site. This finding puts in doubt the specificity of staining by various FLICAs with regard to individual caspases and shows the need for an extreme care in the interpretation of data obtained using these labels.
Subject(s)
Amino Acid Chloromethyl Ketones/analysis , Amino Acid Chloromethyl Ketones/pharmacology , Apoptosis , Caspase Inhibitors , Fluorescent Dyes/analysis , Staining and Labeling , Amino Acid Chloromethyl Ketones/metabolism , Binding Sites , Cell Line, Tumor , Flow Cytometry , Fluorescent Dyes/metabolism , Humans , Kinetics , Propidium , Protein Processing, Post-Translational/drug effects , Protein Subunits/metabolism , Reproducibility of Results , Sensitivity and Specificity , Substrate SpecificityABSTRACT
Characterizing components eluting from a HPLC column is enhanced when multiple detectors are incorporated in-line. The performance of a system consisting of a combination of two detectors-electrospray ionization mass spectrometry (and tandem mass spectrometry) and radioactivity monitoring, following gradient separation with a 250 x 2.1 mm I.D. (Vydac Protein and Peptide C18, 5 microns, 300 A) column-is evaluated with respect to chromatographic integrity and detection. The HPLC effluent was split (8:1) and a post-column make-up solvent was added to flow directed towards the radioactivity detector containing a solid glass cell. Trifluoracetic acid (0.1%) was added to the make-up flow solvent to prevent silanol interactions from degrading the profile displayed in the 14C trace. A 14C chromatographic peak representing 550 dpm was detected with signal-to-noise ratio of 3. This system was used for rapidly characterizing the biliary metabolites of an arginine fluoroalkyl ketone analog of D-MePhe-Pro-Arg, a potent thrombin inhibitor currently being evaluated as a drug candidate. These metabolites are shown to comprise of mono- and dihydroxylated drug as well as a reduced ketone form of the drug. Combining the radioactivity monitor in-line with the mass spectrometer ensured that all of the major metabolites (as evident from the 14C profile) were characterized by mass spectrometry.
