zymografi

Zymography, or zymografi in Swedish, is a gel-based electrophoretic technique used to detect enzyme activity. It combines separation of proteins by polyacrylamide gel electrophoresis with in-gel substrate digestion, producing clear zones where enzymatic activity has occurred. The method is especially used to assess proteases, notably matrix metalloproteinases (MMPs), but variants exist for other enzyme classes.

Principle and variants: In gelatin zymography, samples are subjected to electrophoresis in a polyacrylamide gel containing gelatin or another substrate. After separation, the gel is incubated in a renaturation buffer to remove SDS and restore enzyme activity, followed by incubation in an appropriate buffer to allow substrate degradation. Active enzymes digest the substrate, and the gel is stained so that areas of clearance appear as bands corresponding to the proteases’ molecular weights. Substrate choice defines the enzyme class detected; reverse zymography can be used to detect inhibitors, and in situ zymography allows activity visualization in tissue sections.

Procedure and interpretation: Zymography is typically semi-quantitative. Densitometric analysis of band intensity can estimate relative enzyme activity, often with comparison to standards. Specificity is inferred by inhibition tests (e.g., metalloprotease inhibitors) and by matching band positions to known enzyme standards. The technique requires careful control of sample handling, renaturation conditions, and substrate integrity.

Applications and limitations: Zymography is widely employed in basic and translational research to study protease activity in cancer, fibrosis, cardiovascular disease, and inflammation. It provides functional information complementary to expression analyses but has limitations, including substrate dependence, semi-quantitative output, and potential underrepresentation of enzymes that require special cofactors or non-denaturing conditions. It is primarily a research tool rather than a routine clinical assay.