ImmunoChemistry Technologies’ serine protease inhibitor assays enable researchers to detect intracellular chymotrypsin-like serine protease activity in vitro without lysing the cell. The reagents are non-cytotoxic green or red fluorescent inhibitors that covalently bind with active serine protease enzymes.
It is a powerful method to assess the intracellular levels of chymotrypsin-like serine protease activity in vitro. Just add the serine protease inhibitor probe directly to the cell culture media, incubate, and wash. Because these inhibitors are cell-permeant, they will efficiently diffuse in and out of all living cells. If there is an active chymotrypsin-like enzyme inside the cell, it will covalently bind with the serine protease inhibitor and retain the green or red fluorescent signal within the cell. Cells containing lower concentrations of chymotrypsin-like enzyme activity will retain a lower level of fluorescence compared to cells containing higher concentrations of this effector enzyme. There is no interference from pro-enzymes nor inactive forms of the enzymes. If the treatment is activating chymotrypsin-like serine proteases, positive cells will fluoresce brighter than the normal baseline negative cells, thus enabling researchers to clearly differentiate the populations.
Serine proteases are a family of proteolytic enzymes defined by the presence of a serine residue at the active center of the enzyme, which participates in the formation of an intermediate ester to transiently form an acyl-enzyme complex. The most characterized enzymes of this type are trypsin and chymotrypsin. All living cells have a base level of chymotrypsin- like enzymatic activity which will vary with the physiological state of the cell as well as by cell type. Activated serine proteases play major roles in several different functions including: apoptosis; markers of tumor malignancy; diagnostic and prognostic indicators of breast carcinomas and neck and head carcinomas. Serine protease activity is also altered in a variety of other cell-mediated diseases related to transplant rejection and infections.
Because of their supporting role in the apoptotic process, serine protease activity will be greater in apoptotic cell populations compared to healthy cells of the same cell type. Activation of caspases is upstream and likely a prerequisite for activation of serine proteases. Using serine protease inhibitor probes in combination with FLICA® allows researchers to discriminate serine protease activity from caspase activity in the same cell.
Tightly controlled proteolysis is a defining feature of apoptosis. Caspases are critical in this regard but there are also significant roles for non-caspase proteases. These proteases may complement or accelerate caspase-mediated cell death or they may mediate other forms of caspase-independent cell death. Inhibitors of serine proteases can delay cell death in HL-60 cells. Staurosporine activates serine-protease-dependent cell death independently of, but in parallel with, caspase controlled systems in HL-60 cells. A chymotrypsin-like protease is activated during staurosporine-induced apoptosis and appears to be responsible for specific events downstream of mitochondrial disruption in HL-60 cells. Features of the serine protease-mediated cell-death system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease, and DNA degradation.
Involvement of serine proteases in apoptosis has been mostly studied by observing whether apoptotic events can be prevented by specific inhibitors of these enzymes. Fragmentation of DNA in HL-60 cells treated with DNA topoisomerase inhibitors to induce apoptosis was prevented by the use of an irreversible serine protease inhibitor, such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), which inhibits chymotrypsin. The same inhibitor also inhibited nuclear fragmentation as well as fragmentation of DNA in other cell types, including thymocytes treated with the corticosteroid prednisolone.
Our serine protease inhibitor probes consist of fluorochrome-labeled analogs of the first serine protease inhibitor, tosyl-phenylalanylchloromethyl ketone (TPCK). They are labeled with either a green carboxyfluorescein (FAM) or red sulforhodamine 101 (also known as Texas Red™) fluorochrome, and are available with either a chloromethyl ketone (CMK) or diphenyl 1-(N-peptidylamino) alkane- phosphonate (DAP) reactive-group containing compound. The CMK and DAP reactive groups target the catalytic sites of serine proteases, thus facilitating the covalent labeling of intracellular chymotrypsin-like protease enzymes. CMK-based inhibitors irreversibly alkylate the active site histidine residue of serine proteases, while DAP-based inhibitors irreversibly phosphonylate the active site serine residue to give rise to their respective stable inhibitor-enzyme complexes. Serine protease inhibitor probes include: FAM-phenylalanine-CMK (FFCK); FAM-spacer-phenylalanine-CMK (FSFCK); SR101-phenylalanine-CMK (SFCK); FAM-leucine-CMK (FLCK); FAM-spacer- leucine-CMK (FSLCK); SR101-leucine-CMK (SLCK); FAM-phenylalanine-DAP (FFDAP); FAM-leucine-DAP (FLDAP). FFCK with Phe moiety is expected to inhibit chymotrypsin. FLCK with Leu moiety should have a preference to chymotrypsin C.
Cells labeled with FAM-probes can be counter-stained with other reagents such as the red vital stains Propidium Iodide (included in FAM-probe kits) and 7-AAD (Catalog #6163) to identify necrotic cells. Nuclear morphology may be concurrently observed using Hoechst 33342, a blue DNA binding dye (included in all probe kits). Cells can be viewed directly through a fluorescence microscope, or the fluorescence intensity can be quantified using a flow cytometer or fluorescence plate reader. FAM-probes excite at 488-492 nm and emits at 515- 535 nm. SR101-probes excite at 586 nm and emits at 605 nm.
- Prepare samples and controls
- Dilute cellular wash buffer 1:10 with diH2O
- Reconstitute FLISP with 50 µL DMSO
- Dilute FLISP 1:5 by adding 200 µL PBS
- Add 10 µL FLISP to each sample (~500 µL aliquot of cultured cells)
- Incubate ~ 1 hour.
- Remove media and wash cells: add wash buffer and spin cells (twice); or add fresh media and incubate 1 hour
- If desired, label with additional stains, such as Hoechst, PI, or an antibody
- If desired, fix or embed cells
- Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLISP excites at 488 nm and emits at 520 nm.