Matrix metalloproteinases (MMPs)

Available QuickZyme MMP activity assay kits:

Matrix metalloproteinases (MMPs) are the only enzymes that degrade collagen, and thus are involved in many physiologic processes​ including wound healing​ and embryonic development. Due to differences in protein domains, MMPs are classified as archetypal MMPs (MMP-1,-3,-8,-10,-12,-13,-19,-20, -27), gelatinases (MMP-2,-9), matrilysins (MMP-7,-26), and convertase-activatable MMPs (MMP-11, -14, -15, -16, -17, -21, -23B, -24, -25, -28). Dysregulated expression, aberrant activity, or altered degradation of extracellular matrix (ECM) components, including MMPs, underlies many disease states:

  • Cancer (matrix degradation, cell invasion and migration, angiogenesis, apoptosis, metastasis)​
  • Inflammatory diseases (gut, joints)​
  • Vascular diseases​
  • CNS diseases​
  • Infection (bacterial, virus)​
  • Sepsis​
  • Fibrosis​

MMP activity is analyzed as a biomarker in research applications investigating these disease states. MMP-2​ activity has been examined in the serum of subjects with COPD, bladder cancer, colorectal cancer, and pre-eclampsia​. MMP-7 activity has proven to be a useful biomarker in breast cancer, colorectal cancer, IPF, kidney and lung fibrosis studies. MMP-8​ has been mainly analyzed in periodontitis, gastric cancer, and colorectal cancer research. MMP-9​ activity is used as a biomarker in studies of cardiac remodeling, colorectal cancer, squamous cell carcinoma, bladder cancer, early kidney damage, ocular inflammation (tear fluid), and pre-eclampsia.

Under normal conditions, MMPs are regulated at the levels of transcription, proenzyme activation, localization, and enzymatic activity. MMP activity is inhibited by endogenous inhibitors known as tissue inhibitors of metalloproteinases (TIMPs). MMPs and TIMPs normally exist in a delicate balance, but when the activity of MMPs outweighs the inhibitory functionality of TIMPs within a tissue, ECM breakdown occurs. Thus, TIMPs are also the focus of many research studies and are used as biomarkers for many of the same disease states as MMPs. Side note:  TIMPs are now clinically used in fibrosis tests, like the Enhanced Liver Fibrosis (ELF™) test for the identification of NASH subjects with advanced fibrosis.

The enzymatic activities of MMPs are important to understand, but have been difficult to study due to complexity and lack of sensitive and specific tools to study MMPs in biological samples. While collagen degradation assays are a direct readout on general MMP activity, they do not indicate the activity of specific MMPs. ​Quenched fluorescent (FRET) peptides are effective at evaluating protease kinetics but require purified enzymes and do not indicate MMP specificity in biological samples. Zymography is not high throughput and can activate inactive proteases, further skewing the limited semi-quantitative outputs.

To improve technical specificity, immunocapture activity assays have been developed to measure MMP-specific activity. This technology utilizes an antibody to capture a specific MMP. Next, the proteolytic activity is measured with chromogenic or fluorogenic peptide substrates​. Immunocapture activity methods have proven to be the best assays for assessing specific enzymatic activity in biological samples. Immunocapture activity assays are leading to a better understanding of how MMP activities participate in normal biological processes, disease states, and how they can be further leveraged as fluid-based biomarkers in therapeutic and drug development scenarios.

The Quickzyme approach to analyzing MMP activity utilizes immunocapture activity technology that offers many advantages. As the assay name implies, MMPs are captured with a monoclonal antibody, much like the capture of a standard ELISA. This allows specific MMPs to be isolated from biological samples. MMPs require amino acid bonds on flanking sides of target cleavage sites, which makes classic chromogenic peptide substrate set-ups not possible. Instead, the Quickzyme chromogenic activity assays overcome this by utilizing a modified pro-urokinase that is activated following cleavage by MMPs. Modified urokinase design increases assay sensitivity due to the amplification​ of the detection enzyme. The chromogenic readout can be measured over time and offers a wide dynamic range by kinetic read-out, unlike standard antigen ELISAs.

QuickZyme Chromogenic Activity Assay Strategy:

QuickZyme Matrix metalloproteinases MMP Activity Assay Kits

In addition to the existing activity measurement, there is an optional enzyme activation step of the QuickZyme kits that make it possible to detect both active enzymes and pro-enzymes, after urokinase activation​. This is an advantage over standard total antigen ELISAs, as the enzymatic specificity of what is being captured by the antibodies in antigen ELISAs is generally not known (pro-form, active form, inhibitor complex, or a combination)​.