Chromogenic substrate for continuous spectrophotometric assay of most matrix metalloproteinases, and TACE (ADAM17). The MMP cleavage site peptide bond is replaced by a thioester bond in this peptide. Hydrolysis of this bond by an MMP produces a sulfhydryl group, which reacts with DTNB [5,5'-dithiobis(2-nitrobenzoic acid), Ellman's Reagent] to form 2-nitro-5-thiobenzoic acid, which can be detected by its absorbance at 412 nm.
Suggested protocol for MMP activity assay with thiopeptide substrate (Prod. No. BML-P125)
Dissolve thiopeptide with DMSO or DMF for a 20-50 mM stock solution. To ensure accurate concentration is achieved, both peptide purity and content need to be taken into account.
Here is an example calculating the amount of DMSO needed to dissolve 5 mg BML-P125 to 50mM, when its purity is 97% and content is 93.5%:
(mol/655.9g)x(1x103 mmol/mol)x(L/50mmol)x(1x106µl/L)x(g/1x103mg)x(0.935x5mg)x(0.97)=138.3µl DMSO.
Store at -20°C in aliquots.
Prepare 10X assay buffer:
500mM HEPES
100mM CaCl2
0.5% Brij-35
10mM DTNB [5,5’-dithiobis(2-nitrobenzoic acid)]
pH to 7.0
Store at 4°C in a dark container.
For MMP-2 add 100mM ZnCl2to the 10X buffer, and substitute 500mM MOPS for HEPES. For MMP-3, substitute 500mM MES (2-[N-morpholino]ethane-sulfonic acid) for HEPES and pH to 6.0.
Because optimal amounts of thiopeptide and MMP will vary, it is best to initially use a range of both. Suggested concentration ranges are 0-500mM thiopeptide, and 0-50nM MMP. In the end, initial velocity should be linear with respect to enzyme concentration, and the peptide must be at a saturating concentration for endpoint assays, or well below Kmfor continuous assays. Note: If APMA has been used to activate a pro-MMP, it must be removed (with a G-25 column), as it will react with DTNB. Similarly, DTT, TCEP, and b-mercaptoethanol will also interfere with the assay; these can be dialyzed out.
Reactions (diluted assay buffer, enzyme, and substrate) are monitored in a spectrophotometer at 412nm at 25-37°C. Either cuvette or microplate (using flat-bottomed microplates) format can used. The reaction can be stopped with 50mM EDTA if desired. If MMP inhibitors are being used, incubate MMP with inhibitor for one hour prior to assay. Do not use thiol inhibitors with this substrate; they too will react with DTNB.
NOTE: This protocol serves as a guide only. Exact assay conditions must be determined by the user.
Related products also available from Enzo Lifesciences include fluorogenic MMP substrates, recombinant and purified MMPs, MMP inhibitors, and MMP inhibitor screening kits.
Regulatory Status:
RUO - Research Use Only
Product Literature References
Designed Loop Extension Followed by Combinatorial Screening Confers High Specificity to a Broad Matrix Metalloproteinase Inhibitor: A. Bonadipo, et al.; J. Mol. Biol. 435, 168095 (2023), Abstract;
Utilizing genetic code expansion to modify N-TIMP2 specificity towards MMP-2, MMP-9, and MMP-14: H. Hayun, et al.; Res. Sq. (2023), Abstract;
Toxicological effects of NCKU-21, a phenanthrene derivative, on cell growth and migration of A549 and CL1-5 human lung adenocarcinoma cells: H.F. Liao, et al.; PLoS One 12, e0185021 (2017), Abstract; Full Text
Design, synthesis and evaluation of novel metalloproteinase inhibitors based on L-tyrosine scaffold: X.C. Cheng, et al.; Bioorg. Med. Chem. 20, 5738 (2012), Abstract;
Identifying Chelators for Metalloprotein Inhibitors Using a Fragment-Based Approach: J.A. Jacobsen, et al.; J. Med. Chem. 54, 591 (2011), Application(s): Colorimetric Assay, Abstract; Full Text
Cleavage site specificity and conformational selection in type I collagen degradation: R. Salsas-Escat, et al.; Biochemistry 49, 4147 (2010), Application(s): Assay with human collagen cells, Abstract;
The effect of a hydroxamic acid-containing polymer on active matrix metalloproteinases: G.A. Skarja, et al.; Biomaterials 30, 1890 (2009), Abstract;
Produced in E. coli. Active Matrix Metalloproteinase-3 (MMP-3, stromelysin-1, transin) catalytic domain from human cDNA. The enzyme consists of the catalytic domain of human MMP-3 (Phe100-Thr272, NM_002422) with a C-terminal purification tag., ≥95% (SDS-PAGE) | Print as PDF
