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Collagenase

Cat# 5002 - Recombinant Human MMP-13 (Truncated) - is on back order until future notice. Our apologies for the inconvenience.

Collagenases are members of the matrix metalloproteinase (MMP) family, a group of proteinases that that are involved in the physiological and pathological turnover of the extracellular matrix (ECM) and connective tissues. MMPs can be grouped based on their reported substrate specificities (collagenases, gelatinases, stromelysins) and structures (membrane-type MMPs). Collagenases (MMP-1, MMP-8, MMP-13) can specifically degrade native fibrllar collagen (types I, II, III, VII, and X) in their stereotypical triple helix domain, as well as denatured collagen (gelatin). However, substrate specificity varies among collagenases. Collagenase expression is typically low in healthy tissue, but is elevated in inflammatory conditions, malignant tissue, and other pathological conditions that produce a pro-inflammatory cytokine milieu. Therefore, collagenases are an intriguing therapeutic target for diseases affecting cartilaginous tissues.

To study proteinase activity against native form type I collagen and type II collagen substrates, Chondrex, Inc. provides Rapid Collagenase Activity Assay Kits, as well as Bacterial Collagenase Activity Assay Kits. By using native form collagen as a substrate, these assays can preclude enzymatic activity by other MMPs as only collagenases can cleave native form, fibrillar collagen. Additionally, the MMP-13 Inhibitor Activity Assay Kit is suitable for evaluating the efficacy of MMP-13 inhibitors. 

Chondrex, Inc. also provides full length, latent form Recombinant Human MMP-8 (Cat# 5001), as well as a truncated Recombinant Human MMP-13 (Cat# 5002) that are ideal as references for collagenase activity assays using type I collagen (Cat# 4001) and type II collagen (Cat# 4002) substrates.
To learn more about our various Collagenase/MMP Activity Assay Kits, MMP enzymes, and collagenase substrates, please continue reading below.

Collagenase Assay Kits

Product Catalog # Price (USD)
Bacterial Collagenase Assay Kit 3014 385.00
MMP-13 Inhibitor Assay Kit 3003 510.00
Rapid Collagenase Assay Kit with Type I Collagen Substrate 3001 475.00
Rapid Collagenase Assay Kit with Type II Collagen Substrate 3002 475.00

Collagenase Substrates

Product Catalog # Price (USD)
FITC-Labeled Bovine Type I Collagen Substrate 4001 215.00
FITC-Labeled Bovine Type II Collagen Substrate 4002 244.00

Collagenases (MMPs)

Product Catalog # Price (USD)
Recombinant Human MMP-13 (Truncated) 5002 275.00
Recombinant Human MMP-8 (Full length, Latent Form) 5001 275.00

Rapid Collagenase Activity Assay Kits

The Rapid Collagenase Activity Assay Kits utilize a recombinant human neutrophil pro-collagenase (MMP-8) as a reference collagenase, as well as two types of collagenase activators (APMA and Trypsin). Cat# 3001 uses FITC-labeled bovine type I collagen, while Cat# 3002 uses FITC-labeled type II collagen as a collagenase substrate. Using these fluorescent substrates allows for a 10X faster reaction and shorter working time (~2 hours) than with traditional substrate gel analysis. Each MMP categorized as a collagenase can cleave native form, fibrillar collagen. However, they differ in their affinity for collagen types: MMP-1 more readily degrades type III collagen, MMP-8 more readily degrades type I collagen, and MMP-13preferentially degrades type II collagen. Given these differences, it is important to consider substrate specificity of your MMP when selecting an assay for MMP activity.

Figure 1 below provides more information on the assay system used in these kits. Collagenase will cleave collagen at the N-terminal. 

These FITC-collagen substrates, as well as reference MMP enzymes, are available for purchase separately.

Figure 1. General overview of the Rapid Collagenase Activity Assay Kit procedure. For more information, please see the kit protocol

Activating the reference MMP* and sample MMPs is one of the most crucial steps in this assay. APMA is a universal activator of latent collagenases. However, samples may contain collagenase inhibitors (e.g. alpha 2 macroglobulin, TIMPs) that could prevent reverse APMA activation. Therefore, for these samples, trypsin is also used as an activator. For more information on sample preparation for the collagenase activity assays, please contact us. 

Bacterial Collagenase Activity Assay Kit

Much of what we know about bacterial collagenases is derived from experiments using the first collagenase discovered (of either bacterial or mammalian origin) from Clostridium hisolyticum. Based on these studies, bacterial collagenases were separated into two classes based on their reactivities. Class I collagenases have strong activity against collagen and weak activity towards synthetic molecules. Class II collagenases react strongly with synthetic peptides, but weakly with collagen. However, several other types of bacterial collagenases have been identified since these classifications were made. Pathologically, bacterial collagenases are important virulence factors for numerous pathogenic bacteria and are chiefly responsible for degrading host ECM and providing a niche for bacterial growth and proliferation (1). Interestingly, Clostridium histolyticum collagenase has been used therapeutically to treat patients with Dupuytren’s diseases and Peyronie’s disease (2,3).

The Bacterial Collagenase Activity Assay Kit utilizes a Clostridium histolyticum collagenase as a reference with FITC-labeled soluble bovine type I collagen substrate. This assay system provides faster reaction time and higher sensitivity than substrate gel analysis and radiolabeled collagen-based assays. Approximately 100 samples of collagenase activity assay and/or collagenase inhibitor assays can be run with this kit.
This kit is similar to the Rapid Collagenase Activity Assay Kits for mammalian MMPs (collagenases), however there are a couple of key differences. Unlike mammalian MMPs, bacterial collagenases will completely degrade collagen into denatured peptide fragments. Thus, elastase digestion is necessary for this assay. Additionally, bacterial assays are generally found in their active form already, therefore no activation step is necessary. 
The Bacterial Collagenase Activity Assay Kit is ideal for assaying class I collagenase activity, as well as evaluating class I collagenase inhibitors. Please see the kit protocol for more information on the different protocols. 

Calculation of enzyme activities

The collagenase activity kits include reference collagenases, however enzyme activities in samples must not be calculated using a standard curve as one would in an ELISA. Instead, collagenase activity must be calculated as units/min/ml to allow for a proper comparison between standards and samples (Figure 2).

MMP-8 activity curve (micrograms collagen/minutes) using FITC-labeled type II collagen as a substrate

Figure 2. Example of MMP-8 activity curve. A) MMP-8 activity dose response using FITC-type I collagen as a substrate - Various amounts (2.5 – 10 µl) of reference MMP-8 (100 units/ml) were reacted with 100 µg of FITC-type I collagen at 35°C for 60 minutes. The mixtures were further incubated at 35°C for 10 minutes after adding 10 µl of Enhancer. B) MMP-8 activity time couse using FITC-type I collagen as a substrate - 2.5 ml of MMP-8 (100 units/ml) was reacted with 100 µg of FITC-type I collagen at 35°C for 90 minutes.

One unit of collagenolytic activity is defined as the cleavage of 1 µg of collagen per minute (1 collagenolytic unit = 1 µg/minutes). Since this kit uses 100 µg of collagen as a substrate, collagenolytic activity can be calculated using the following equation:

Figure 3. Equation to calculate collagenolytic activity in Chondrex, Inc.’s Collagenase Activity Assay Kits. FIsample: Fluorescent Intensity of sample; FIblank: Fluorescent Intensity of blank FIcontrol: Fluorescent Intensity of control

This kit’s assay sensitivity is heavily influenced by the reaction (incubation) time. A longer incubation time will increase assay sensitivity. However excessive incubation will increase background signal due to degradation or denaturation of collagen fragments by other proteinases. In addition, insufficient reaction time (10 minutes or less) may affect assay reproducibility. The recommended incubation time is one hour; however the reaction time must be optimized for your samples. The assay sensitivity can be determined by comparison of fluorescence intensity (FI) of samples and FI of blanks. For accurate assay results, the FI of samples should be at least 1.5x higher than the FI of blanks. For instance, if the blank FI is 1000, the sample FIs must be higher than 1500 for accurate assay results.  

The assay temperature will also affect the assay sensitivity. Once collagen is heat denatured (40°C for native collagen, 35°C for collagenase cleaved collagen) the sample results may be overestimated due to elastase digestion of denatured collagen. Furthermore, the enzyme specificity for the collagen substrate is an important consideration when choosing a substrate. 

FITC-Labeled Type I and Type II Collagen

FITC-labeled bovine type I and type II collagen (conjugated at a 1:1 ratio) are excellent substrates for examining collagenase activity. To minimize background values in MMP (collagenase) assays, these labeled substrates have been prepared telopeptide free. Additionally, FITC that is non-specifically bound to collagen has been removed by ion-exchange chromatography.

FITC-labeled collagen can also be used to create scaffolds for primary cell cultures or establish cell cultures. If used in this way, collagenase activity from cells can be determined by measuring the FI of degraded FITC-collagen in culture supernatants (520 nm emission/490 nm excitation). However, proteins and dyes present in culture medium may result in quenching of FI. Optimizing the assay and culture conditions can help ensure consistent and reliable results.

For this reason, Chondrex, Inc. suggests to use a high concentration of FITC-collagen when creating cell culture scaffolds. Please contact us for more information.  

Chondrex, Inc. also offers many other grades of purified collagen for a variety of experimental purposes. 

MMP-13 Inhibitor Assay Kit

MMP-13 (Collagenase 3) is a matrix metalloproteinase originally identified in breast carcinomas (4). Subsequently, MMP mRNA assays have shown increased expression levels of MMP-13 in osteoarthritis (OA) chondrocytes (5-7), as well as in a variety of malignant tumor types (8). Physiologically, MMP-13 functions primarily during embryonic development (9,10) and wound healing (11,12). MMP-13 production in chondrocytes and synoviocytes has been observed to be up-regulated by inflammatory mediators such as Interleukin-17 (IL-17), IL-1, Tumor Necrosis Factor Alpha (TNF-α), and Oncostatin M (13-15). Naturally occurring Tissue Inhibitors of Metalloproteinases (TIMPs) also play key roles in the regulation of MMP activity (16).

Given MMP-13’s preference to degrade type II collagen, it has been associated with cartilage destruction in rheumatoid arthritis (RA) and OA. Therefore, several MMP-13 inhibitors have been tested as therapeutic compounds in the mouse collagen induced arthritis model (17). Indeed, several factitious MMP inhibitors (primarily zinc chelators like batimastat and marimastat that block the catalyitc site of MMPs) have been developed and extensively studied as therapeutic agents in a variety of diseases. However, they all failed during clinical trials due to the severe and systemic side effects (18). Recently, new classes of more selective MMP inhibitors (e.g. monoclonal antibody-based inhibitors and engineered proteins) have gained interest as therapeutics options in cancer and OA. These new synthetic MMP inhibitors that can target specific MMPs could prove to be useful tools in treating diseases where tissue remodeling and/or connective tissue degradation are a distinguishing feature. 

Chondrex, Inc.’s MMP-13 Inhibitor Assay Kit provides an easy way to screen for and evaluate the efficacy of experimental MMP-13 and other MMP inhibitors. Using a truncated, recombinant human MMP-13 (rhMMP13) and a proteinase inhibitor as references, you can monitor the cleavage of the synthetic, fluorogenic peptide substrate*. Simply use any experimental inhibitor compound of interest in place of the proteinase inhibitor (Cat# 30046) provided with the kit to evaluate the inhibitory potential of the compound. 

*Substrate does not have collagen triple helix and therefore is susceptible to non-collagenolytic proteinases.

Figure 4 - Dendritic cells producing collagenase:  Rat synovial cell stimulated by neutrophil extract containing IL-1 triggers the morphological change of fibroblasts into dendritic cells.

Recombinant Human MMP-8 (Collagenase 2)

MMP-8 (also known as neutrophil elastase) is a collagenase released by activated neutrophils that preferentially degrades type I collagen. MMP-8 proteolytic activity is inhibited by both TIMP-1 and TIMP-2. Unlike many other MMPs, MMP- 8’s function appears to suppress tumor metastasis (19,20) as well as experimental arthritis models (21). Chondrex, Inc.’s Recombinant Human MMP-8 is a useful reference collagenase when analyzing MMP dynamics in both physiological and pathological conditions. 

When assaying for collagenase activity or evaluating MMP/collagenase inhibitors (such as TIMPS), one of the most critical steps is activation of latent collagenase enzymes. Chondrex, Inc.’s Recombinant Human MMP-8 is a full-length, latent collagenase that can be helpful for this purpose. Additional, Cat# 3001 can be activated using APMA, making it useful to evaluate MMP (collagenase) inhibitors as well. 

Recombinant Human MMP-13 (Collagenase 3), Truncated

MMP-13 serves several physiological roles (bone metabolism, wound healing) as well as roles in several pathological conditions (tumor invasion & metastasis (22), osteoarthritis (23)). The active form of recombinant human pro-collagenase MMP-13 (truncated from the C-terminal) is available for studying inhibitors by using a fluorogenic synthetic peptide as a substrate. However, it is important to note that this truncated form of MMP-13 cannot digest native collagen as it does not contain a collagen binding motif.

Trypsin Activity Assay Kit

Trypsin, produced by the pancreas, plays a key role in facilitating protein digestion and absorbance of nutrients in the small intestine. It also helps regulate the gastrointestinal immune response by controlling microbicide concentrations in the intestinal lumen and maintaining the integrity of the epithelial barrier (24,25). Due to these capabilities, trypsin is widely used in protein or peptide-related research for synthesizing and sequencing peptides, maintaining cultured cells, and digesting proteins (26). 

Chondrex, Inc. provides a chromogenic Trypsin Activity Assay Kit which uses a Boc-Gln-Ala-Arg-pNA substrate to measure trypsin-like enzyme activity in as little as 15 minutes. The trypsin cleaves the carbonyl group in the Arg of the substrate, liberating p-nitroanilid (pNA) which produces a yellow color and can be quantified using optical density (27). This kit works for trypsin and any proteinase/peptidase which cleaves the substrate.

 

References

  1. A. S. Duarte, A. Correia, A. C. Esteves, Bacterial collagenases - A review. Crit Rev Microbiol 42, 106-126 (2016).
  2. R. De Vitis et al., Seven-year clinical outcomes after collagenase injection in patients with Dupuytren's disease: A prospective study. J Orthop 21, 218-222 (2020).
  3. S. C. Honig, Intralesional collagenase in the treatment of Peyronie's disease. Ther Adv Urol 6, 47-53 (2014).
  4. J. M. Freije et al., Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J Biol Chem 269, 16766-16773 (1994).
  5. M. B. Goldring et al., Roles of inflammatory and anabolic cytokines in cartilage metabolism: signals and multiple effectors converge upon MMP-13 regulation in osteoarthritis. Eur Cell Mater 21, 202-220 (2011).
  6. M. Wang et al., MMP13 is a critical target gene during the progression of osteoarthritis. Arthritis Res Ther 15, R5 (2013).
  7. S. Kamekura et al., Osteoarthritis development in novel experimental mouse models induced by knee joint instability. Osteoarthritis Cartilage 13, 632-641 (2005).
  8. M. Balbín et al., Expression and regulation of collagenase-3 (MMP-13) in human malignant tumors. APMIS 107, 45-53 (1999).
  9. M. Inada et al., Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification. Proc Natl Acad Sci USA 101, 17192-17197 (2004).
  10. N. Johansson et al., Collagenase-3 (MMP-13) is expressed by hypertrophic chondrocytes, periosteal cells, and osteoblasts during human fetal bone development. Dev Dyn 208, 387-397 (1997).
  11. M. Toriseva et al., MMP-13 regulates growth of wound granulation tissue and modulates gene expression signatures involved in inflammation, proteolysis, and cell viability. PLoS One 7, e42596 (2012).
  12. B. Hartenstein et al., Epidermal development and wound healing in matrix metalloproteinase 13-deficient mice. J Invest Dermatol 126, 486-496 (2006).
  13. P. J. Koshy et al., Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines. Ann Rheum Dis 61, 704-713 (2002).
  14. A. D. Rowan, W. Hui, T. E. Cawston, C. D. Richards, Adenoviral gene transfer of interleukin-1 in combination with oncostatin M induces significant joint damage in a murine model. Am J Pathol 162, 1975-1984 (2003).
  15. W. Hui, A. D. Rowan, C. D. Richards, T. E. Cawston, Oncostatin M in combination with tumor necrosis factor alpha induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo. Arthritis Rheum 48, 3404-3418 (2003).
  16. K. Brew, H. Nagase, The tissue inhibitors of metalloproteinases (TIMPs): an ancient family with structural and functional diversity. Biochim Biophys Acta 1803, 55-71 (2010).
  17. A. Jüngel et al., Effect of the oral application of a highly selective MMP-13 inhibitor in three different animal models of rheumatoid arthritis. Ann Rheum Dis 69, 898-902 (2010).
  18. A. Winer, S. Adams, P. Mignatti, Matrix Metalloproteinase Inhibitors in Cancer Therapy: Turning Past Failures Into Future Successes. Mol Cancer Ther 17, 1147-1155 (2018).
  19. A. Gutiérrez-Fernández et al., Matrix metalloproteinase-8 functions as a metastasis suppressor through modulation of tumor cell adhesion and invasion. Cancer Res 68, 2755-2763 (2008).
  20. J. T. Korpi et al., Collagenase-2 (matrix metalloproteinase-8) plays a protective role in tongue cancer. Br J Cancer 98, 766-775 (2008).
  21. J. H. Cox et al., Matrix metalloproteinase 8 deficiency in mice exacerbates inflammatory arthritis through delayed neutrophil apoptosis and reduced caspase 11 expression. Arthritis Rheum 62, 3645-3655 (2010).
  22. M. F. Leeman, S. Curran, G. I. Murray, The structure, regulation, and function of human matrix metalloproteinase-13. Crit Rev Biochem Mol Biol 37, 149-166 (2002).
  23. V. V. Ravi Kanth, D. Nageshwar Reddy, Role of matrix metalloproteinases in physiological processes & disease. Indian J Med Res 140, 585-587 (2014).
  24. M. Bajaj-Elliott, Trypsin and host defence: a new role for an old enzyme. Gut 52, 166-167 (2003).
  25. D. Ghosh et al., Paneth cell trypsin is the processing enzyme for human defensin-5. Nat Immunol 3, 583-590 (2002).
  26. J. A. Mótyán, F. Tóth, J. TÅ‘zsér, Research applications of proteolytic enzymes in molecular biology. Biomolecules 3, 923-942 (2013).
  27. B. F. Erlanger, N. Kokowsky, W. Cohen, The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95, 271-278 (1961).