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 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.
|Alternative Name:||Neutrophil collagenase, Collagenase-2|
|Applications:||Fluorescent detection, HTS|
|Application Notes:||Designed to screen MMP-8 inhibitors using a quenched fluorogenic peptide.|
|Handling:||Avoid freeze/thaw cycles.|
|Shipping:||Shipped on Dry Ice|
|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
|Scientific Background:||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.|
|Technical Info/Product Notes:||NCBI Reference Sequence: NM_002424|
The OMNIMMP® RED substrate offers key advantages over other MMP substrates.
- 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.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.
- OMNIMMP® RED is avidly cleaved by MMPs, with kcat/Kms in the range of 104-106 M-1sec-1.
- The ultra-strong fluorescence of OMNIMMP® RED allows for substrate concentrations much lower than the Km, a condition generally desirable in inhibitor screening assays.
Product Literature References
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Examined enzyme activity of the recombinant proteins and cleavage products, Abstract
General Literature References
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Chondrocyte matrix metalloproteinase-8. Human articular chrondrocytes express neutrophil collagenase
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Characterization of 58-kilodalton human neutrophil collagenase: comparison with human fibroblast collagenase
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