I am trying to detect MMP activity in BMDC total cell lysates and supernatants. This has proven to be more difficult than I anticipated. I am using fluorescein labeled gelatin (Enzchek Gelatinase/ Collagenase assay kit from molecular probes) as the substrate for detection in my assay. It is very unlikely that there are no MMPs (or other proteases that can degrade gelatin) in my samples. Is there a better substrate for measuring total MMP activity? Are there MMPs that do not degrade collagen?
A number of membrane bound MMPs will not accept gelatin as a substrate. It is also worth noting that MMPs are zymogens that probably need to be activated in vitro to observe activity from cell lysates.
Hi Einar, do you have your cells in serum containing media? Do you wash away the serum media before lysing your cells?
Serum has a ton of proteinase inhibitors. Are the cell supernatants you assay serum free?
I use a colorimetric generic MMP assay from Anaspec to measure total MMP activity as absorbance. It has a chromogenic thiopeptolide substrate. My samples also have very low amount MMP but it works (we modified the kit's protocol actually for our experiment.
Is there a better substrate for measuring total MMP activity?
I have worked mostly on the gelatinases (MMP-2 and some on map-9), and gelatine zymography is an excellent way to get a a qualitative indication of presence of these two. In my opinion - its horrible for quantitative. Its good because of mentioned reasons - its sensitive and with SDS it disrupts the zymogen inactivity enough so that also the inactive zymogen is active in the assay. There are publications describing how (or atleast postulates how) this happens. For a quantitative activity measurement I would use a FRET assay on further purified material (see my publications for instance).
No MMP activity is kind of strange (I am not familiar with your cells sadly) and could be due to mishandling of the sample (so that other enzymes degrade the MMPs in the sample) or large quantities of MMP inhibitors.
I suspect that there are plenty of zymogen protocols that does not work optimal either (potentially with too little time for the enzyme to cleave the gelatine in the gel). I would look at different protocols that lets the enzyme act for a longer time. I could check my own if you like. Why not also double check with western blotting and preferable a polyclonal antibody? (not activity based)
Are there MMPs that do not degrade collagen?
The ability of each enzyme to efficiently hydrolyze the triple helix is varying, and the gelatinases are weaker collagenolytic enzymes than for instance MMP-1. Please remember that native collagen and gelatine, and those used in commercial assays can differ in many ways.
The collagen fibre consists of three α-chains of approximately 1000 residues with repeating Gly- X (often Pro)- Y (often HYP), which adopts a left-handed poly- Pro II like helix. Three of these α-chains intertwine to form a right-handed superhelix, and the structural complexity makes it resistant to proteolysis by most enzymes.
Substrate residues might facilitate local unwinding. Enzymes also need regions outside of the active site for their affinity towards high-molecular-weight substrates, which are sometimes referred to as exosites. The helix needs to be unwound, and work on a catalytically inactive MMP-1 showed that the helicase activity was not depended on the catalytic glutamate. MMP-2 and -9 apparently required their inserts of FnII-like modules to hydrolyze elastin, collagen, and THP mimics of collagens.
MMP-2 has a collagenase activity, and the catalytic and hemopexin domains were shown sufficient to cleave rat type I collagen. A later study found that collagen cleavage was reduced without the fibronectin 15 repeats, and found indications that MMP-14 and MMP-2 bind collagen at a different site than MMP-11 and MMP-8. MMP-9 has also been found to cleave triple helical collagens. The formation of fibers exemplifies that increased structure complexity of the substrate also can lead to substrate specifity.
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