Collagenase

Collagenases, members of the matrix metalloproteinase (MMP) family, are involved in the physiological and pathological turnover of connective tissues. Collagenase production is significantly increased at the sites of inflammation due to the stimulation of pro-inflammatory cytokines. Chondrex provides recombinant human MMP-8 and MMP-13, in addition to collagenase assay kits using FITC-labeled type I collagen and type II collagen, or a fluorogenic peptide as substrates.

Collagenase Assay Kits

Collagenase Substrates

Collagenases (MMPs)

Rapid Collagenase Assay Kit (I) and (II)

The Collagenase Assay Kits (catalog # 3001 or 3002) contain all the necessary reagents to assay collagenase activity including recombinant human neutraphil pro-collagenase (MMP-8) as a reference and two types of collagenase activators. Unlike antibody-based ELISA kits that only detect the presence of enzyme proteins, these kits are designed to assay collagenase activity.  The assay uses FITC-labeled soluble bovine type I collagen (catalog # 3001) or bovine type II collagen (catalog # 3002) as a substrate, and only requires two hours to complete. The enzyme reaction is ten times faster than substrate gel analysis and is much more sensitive than assays using radiolabeled collagen. Each kit includes FITC-labeled collagen to perform approximately 100 assays.

MMP13 Inhibitor Assay Kit

MMP-13 (Collagenase 3) is a newly discovered matrix metalloproteinase found in various tissues such as malignant tumors, osteoarthritic cartilage, rheumatoid synovium, and wounds.  MMP-13 production in chondrocytes and synoviocytes is up-regulated by stimulation with inflammatory mediators such as IL-1, TNF, and retinoic acid.   

Inhibitors of MMPs may be useful therapeutics to prevent metastasis of certain cancer cells and tissue damage in inflammatory diseases.  This MMP-13 Inhibitor Assay Kit contains a truncated, rhMMP-13, which cleaves a fluorogenic peptide substrate and is ideal for assessing inhibitors of MMP-13 and related enzymes.  MMP-13 inhibitors can be easily screened and evaluated by simply adding potential inhibitor compounds to the activated recombinant human MMP-13 (rhMMP-13).

FITC-Labeled Type I and Type II Collagen

FITC-labeled bovine type I and type II collagen are excellent substrates for examining collagenase activity.  To minimize background values in collagenase assays, these labeled substrates are telo-peptide free, and FITC which non-specifically bound to collagen has been removed by ion-exchange chromatography.  FITC-labeled collagen can also be used as a substrate for cell culture.  Collagen degradation products in the culture supernatants can be directly determined by measuring the fluorescence at 520 nm (emission)/490 nm (excitation).

Recombinant Human MMP-8 (Collagenase 2)

Recombinant latent human neutrophil procollagenase (MMP-8) can be used for various purposes such as a reference standard for assaying collagenase activity.  The most critical process in assaying for collagenase is the activation of inactive forms of collagenase.  Since the activation method of collagenase depends on the sources and types of enzyme and selection of activators, it is important to have an inactive latent reference collagenase.  Furthermore, activated MMP-8 can be used for testing inhibitors.

Recombinant Human MMP-13 (Collagenase 3), Truncated

The active form of recombinant human pro-collagenase MMP-13 (truncated form) is available for studying inhibitors using a fluorogenic synthetic peptide as a substrate. 

NOTE: This truncated enzyme is not capable of digesting native collagen.

Figure 1 - Dendritic cells producing collagenase:  Rat synovial cell stimulated by neutrophil extract containing IL-1 triggers the morphological change of fibroblast into dendritic cells.

More Information

MMP-13 (Collagease 3) has been shown to degrade type I and type II collagen, and the degradation of type II collagen occurs approximately ten times faster than that of type I collagen.The typical 3/4 and 1/4 fragments of collagen are produced as with MMP-1.  However, MMP-13 also generates a second cleavage of type II collagen that removes three amino acids from the amino terminus of the 1/4 fragment.  In addition, MMP-13 degrades aggrecan, the major proteoglycan of cartilage.

Application

The fluorogenic peptide substrate used in this kit does not hold a triple helical structure, and might be susceptible to non-collagenolytic proteinases.  Since it is important to know the total activity of collagenase-like proteinases in specimens before proceeding with further experiments, this kit is useful for assaying total collagenase-like proteinase activity in tissue fluids or extracts and cell culture supernatants.