Objective:

 

To analyse the proteolytic activity in cell extracts.

 

Theory:

 

Zymography is a technique to assess the enzymatic activity of proteins either in situ or by separating them with electrophoresis. The enzyme converts the substrate into a product which is detected by different staining methods. One of the most popular technique is by separating the protein mixture by Polyacrylamide gel electrophoresis in which a  substrate is incorporated within  the polyacrylamide gels. These protein substrates present in the gel are degraded by the proteases present in the sample which are activated during the incubation period.  Staining with Coomassie blue shows the proteolytically cleaved sites as white clear bands on a dark blue background. Necessary precautions are taken to prevent the enzyme from denaturation during the process. Hence SDS is generally avoided. But in gelatinase zymography, SDS is used to activate the gelatinases.

 

Gelatin Zymography:

 

Gelatin Zymography is used to detect gelatinase activity, specially MMP-2 and -9. MMP-2 and -9 remain inactive while they are with their pro-domains. They need proteolytic processing or denaturation to get activated. SDS in SDS-PAGE can do that activation by denaturing the MMP_2 and -9. MMP-2 (gelatinase A, 72 kD) and MMP-9 (gelatinase B, 92 kD) can be detected on gelatin zymograms as two-three white bands (pro and active forms) after staining with Coomassie Blue staining.

 

Mechanism of activation of MMP-2:

 

In the  propeptide  domain  of  the MMPs,  a  cysteine  residue  (Cys73)  is  present. In  the  catalytic domain, an active Zn2+ site is  present, which  forms a  bond  with  the  cysteine residue. When  this Cys73-Zn2+  bond  is  intact,  the MMP  is  inactive.

 

 

When the bond between the zn²⁺ present in the active site and the cysteine residue is broken, the  MMPs will get activated. This  mechanism has  been  referred  to  as the  “cysteine  switch” . A water molecule  then binds to the Zn2+ ion and  replaces  the  cysteine residue  after  the  dissociation . Thus an intermediate active enzyme  results .  After subsequent proteolysis occurs then an active MMP  is resulted.

 

 

 

Applications:

 

To visualize the activity of gelatinases like MMP-2, -9 and quantitate the relative amount of pro and active forms of the enzymes .

 

     

Note:
 

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