Quantitative zymography: detection of picogram quantities of gelatinases

Abstract

Zymography is an electrophoretic technique used to identify proteolytic activity in enzymes separated in polyacrylamide gels under nonreducing conditions. It has been used extensively in the qualitative evaluation of proteases present in tumors and cell culture conditioned media. Using commercially available precast gels and a modern image analysis system, we have evaluated zymography as a quantitative technique. The degree of digestion of gelatin within the zymogram by purified gelatinase A, a matrix metalloprotease, is directly proportional to the amount of enzyme loaded over a 10- to 20-fold range. With an overnight (18 h) digestion period, the linear range of this assay extended from 10 to 120 pg of enzyme. The initial rate of digestion is proportional to the enzyme loading and varying the incubation time results in a shift in the linear range of the assay. Active and latent forms of gelatinase A show the same degree of digestion in this assay system. These results justify the use of zymography in the quantitative assessment of gelatinase activity as well as demonstrate its usefulness as a qualitative technique for the analysis of gelatinase species present.