Description
Description: Detect changes in intracellular chymotrypsin-like enzyme activity in whole living cells with the FLISP, Fluorescent-Labeled Inhibitors of Serine Proteases, product line of assay kits. These kits utilize either a leucine (L) or phenylalanine (F) chymotrypsin-targeting amino acid residue linked on the amino terminus with either a carboxyfluorescein (FAM) or sulforhodamine 101 (SR) fluorescent reporter tag. The carboxyl end of the F or L amino acid residue contains a reactive group that targets the catalytic site of serine proteases, consisting of either a chloromethyl ketone (CMK) or (aminoalkyl) phosphonate diphenyl ester (DAP) reactive group. After quickly penetrating the lipid bilayer membranes of the target cell population, the chymotrypsin-targeting FLISP probes will interact with the active catalytic sites of chymotrypsin-like proteases, quickly forming covalent bonds with either the reactive site histidine (N-H) (with the CMK-type FLISP probes) or the reactive serine OH group (when DAP-containing FLISP probes are utilized). In either case, unbound FLISP reagent is easily removed during the wash step, leaving cells with greater quantities of active chymotrypsin-like enzyme activity with a greater fluorescence potential than cells that did not undergo an upregulation of serine protease activity. FLISP probes bearing FAM reporter dyes are excited at 488 nm and emit in the green wavelength range of 525 nm. The red fluorescence FLISP probes that utilize sulforhodamine 101 as the reporter dye are excited at 590 nm and emit at 620 nm. FLISP probes are cell permeant and non-cytotoxic at the concentrations suggested in the assay protocol. The FLISP FAM-Phe-CMK (FFCK) chymotrypsin enzyme detection assay kits can be used in conjunction with existing apoptosis detection protocols. They have been used successfully in tandem with the SR (red) FLICA caspase detection assay kits to show a parallel upregulation of chymotrypsin-like enzyme activity and caspase activation. Each kit includes the FLISP FFCK reagent, 10x wash buffer for removing excessFLISP probe following the FLISP incubation step, 10X fixative to stabilize cells if next day analysis is preferred, Propidium Iodide vital stain for necrotic cell detection, and Hoechst 33342 dye to visualize nuclear morphology. FLISP FFCK-stained cells may be analyzed using flow cytometry, fluorescence microscopy, and fluorescence plate reader evaluation methodology.
Background: Chymotrypsin like enzymes cleave their substrate proteins at the carboxy terminal end of amino acids containing hydrophobic aliphatic or aromatic R-group side chains such as those found on leucine and phenylalanine amino acid structures [1]. The FFCK FLISP probe, consisting of FAM-F-CMK, is simply a fluorescent-labeled analog of the early chymotrypsin-like enzyme inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) [2]. Early studies using topoisomerase inhibitors in HL-60 cells indicated that TPCK, N-tosyl- L-lysine chloromethyl ketone (TLCK) and other serine protease inhibitors, were able to prevent internucleosomal DNA degradation associated with apoptosis [3]. Dual staining experiments have used this FAM-F-CMK chymotrypsin detection probe and the sulforhodamine (SR)-VAD-FMK FLICA caspase detection probe in the presence or absence of z-VAD-FMK, TPCK, or TLCK caspase or serine protease inhibitors [4-5]. This work demonstrated a significant inhibitory effect of the caspase inhibitor, z-VAD-FMK, on chymotrypsin associated FAM-F-CMK binding in cells. This group also showed that the chymotrypsin-like enzyme inhibitor, TPCK, had a strong suppressive effect on caspase activation and apoptosis induction. In contrast, the trypsin-like enzyme inhibitor, TLCK, had very little effect on caspase activation events as measured by the binding of the red FLICA probe SR-VAD-FMK in camptothecin induced cell populations [5]. The FAM-F-CMK chymotrypsin-like protein affinity label was also utilized to analyze upregulated serine protease activity resulting from staurosporine induction in HL-60 and Jurkat cells [6-7]. In staurosporine-induced Jurkat cells, the FAM-F-CMK specifically labeled an upregulated, constitutively expressed, 60 kDa protein as well as a 50 kDa protein that was only detected in staurosporine-treated Jurkat cells [6]. In a related study using staurosporine-induced HL-60 cells, a 16 kDa protein was detected by the FAM-F-CMK FLISP probe [7]