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Matrix metalloproteinase-8 (MMP-8) fluorometric drug discovery kit, RED

 
BML-AK305-0001 96 wells 500.00 USD
 

Product Specification

Alternative Name:Neutrophil collagenase, Collagenase-2
 
Application:Designed to screen MMP-8 inhibitors using a quenched fluorogenic peptide.
 
Handling:Avoid freeze/thaw cycles.
 
Long Term Storage:-80°C
 
Kit/Set Contains:1 vial MMP-8 enzyme
1 vial Substrate (OmniMMP™ RED)
1 vial 6’-TAMRA calibration standard
1 vial control inhibitor (NNGH)
1 bottle (20 ml) assay buffer
1 black 96-well microplate
Instructions
 
Miscellaneous/General:Matrix metalloproteinase-8 (MMP-8, neutrophil collagenase, collagenase-2) is a member of the MMP family of extracellular proteases. These enzymes play a role in many normal and disease states by virtue of their broad substrate specificities. Targets of MMP-8 include collagen, gelatin, aggrecan, entactin, and α1-proteinase inhibitor. MMP-8 is secreted as a 55-80kDa glycosylated proenzyme (as measured by SDS-PAGE), and activated by cleavage to 46kDa and below. MMP-8 is an important target for inhibitor screening due to its involvement in diseases such as cancer, arthritis, and asthma.
 
The MMP-8 Fluorometric (also known as fluorimetric) Drug Discovery Kit, RED is a complete assay system designed to screen MMP-8 inhibitors using a quenched fluorogenic substrate OmniMMP™ RED: TQ3-GABA-Pro-Cha-Abu-Smc-His-Ala-Dab(6-TAMRA)-Ala-Lys-NH2 [TQ3=quencher; GABA=4-aminobutyric acid; Cha=L-cyclohexylalanine; Abu=2-aminobutyric acid; Smc=S-methyl-L-cysteine; Dab=2,4-diaminobutyric acid; 6-TAMRA=6-tetramethylrhodamine]. TAMRA fluorescence is thoroughly quenched by the TQ3 group until cleavage by MMPs separates the two moieties.
 
The OmniMMP™ RED substrate offers key advantages over other MMP substrates. 1) Emission at the red end of the spectrum (576 nm after excitation at 545 nm) avoids the interference at lower wavelengths often exhibited by screening compounds, and by substances commonly found in biological samples and tissue culture medium. 2) MMP substrate peptides display poor aqueous solubility, often with Kms near their limits of solubility, making enzyme and inhibitor kinetics difficult. MMP Kms for OmniMMP™ RED substrate are below its solubility limit. 3) OmniMMP™ RED is avidly cleaved by MMPs, with kcat/Kms in the range of 104-106 M-1sec-1. 4) The ultra-strong fluorescence of OmniMMP™ RED allows for substrate concentrations much lower than the Km, a condition generally desirable in inhibitor screening assays.
 
The assays are performed in a convenient 96-well microplate format. The kit is useful to screen inhibitors of MMP-8, a potential therapeutic target. The compound NNGH is also included as a prototypic control inhibitor.
 
Background / Technical Information:NCBI Reference Sequence: NM_002424
The OmniMMP™ RED substrate offers key advantages over other MMP substrates. 1) Emission at the red end of the spectrum (576 nm after excitation at 545 nm) avoids the interference at lower wavelengths often exhibited by screening compounds, and by substances commonly found in biological samples and tissue culture medium. 2) MMP substrate peptides display poor aqueous solubility, often with Kms near their limits of solubility, making enzyme and inhibitor kinetics difficult.  MMP Kms for OmniMMP™ RED substrate are below its solubility limit. 3) OmniMMP™ RED is avidly cleaved by MMPs, with kcat/Kms in the range of 104-106 M-1sec-1. 4) The ultra-strong fluorescence of OmniMMP™ RED allows for substrate concentrations much lower than the Km, a condition generally desirable in inhibitor screening assays.
 

General Literature References

Matrix metalloproteinases: regulators of the tumor microenvironment: K. Kessenbrock & Z. Werb; Cell 141, 52 (2010), Abstract;
Updated biological roles for matrix metalloproteinases and new "intracellular" substrates revealed by degradomics : G.S. Butler & C.M. Overall; Biochemistry 48, 10830 (2009), Abstract;
Airway obstruction correlates with collagenase-2 (MMP-8) expression and activation in bronchial asthma: K. Prikk, et al.; Lab. Invest. 82, 1535 (2002), Abstract;
Matrix metalloproteinases: they're not just for matrix anymore!: L.J. McCawley & L.M. Matrisian; Curr. Opin. Cell Biol. 13, 534 (2001), Abstract;
Cleavage of native cartilage aggrecan by neutrophil collagenase (MMP-8) is distinct from endogenous cleavage by aggrecanase: E.C. Arner, et al.; J. Biol. Chem. 272, 9294 (1997), Abstract; Full Text
Discovery of CGS 27023A, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits: L.J. MacPherson, et al.; J. Med. Chem. 40, 2525 (1997), Abstract;
Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline: R. Hanemaaijer, et al.; J. Biol. Chem. 272, 31504 (1997), Abstract; Full Text
Chondrocyte matrix metalloproteinase-8. Human articular chrondrocytes express neutrophil collagenase : A.A. Cole, et al.; J. Biol. Chem. 271, 11023 (1996), Abstract; Full Text
Characterization of 58-kilodalton human neutrophil collagenase: comparison with human fibroblast collagenase: S.K. Mallya, et al.; Biochemistry 29, 10628 (1990), Abstract;

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