45] Zymographic techniques for detection and characterization of microbial proteases

Abstract

We have presented a variety of zymographic techniques for identification and characterization of microbial proteases, using SDS-PAGE and PAGE in nondissociating gels. Techniques are described using copolymerized protein substrates, diffusable protein substrates, protein substrates incorporated into indicator gels, as well as synthetic esterase substrates. When a newly discovered protease is being characterized, it is advisable to try a variety of techniques, both to determine optimal conditions for enzyme detection and to characterize the protease. Zymography is a versatile two-stage technique involving protein separation by electrophoresis followed by detection of proteolytic activity. Each particular combination of protease separation and detection techniques had advantages and limitations. Protease separation by SDS-PAGE has as a limitation the fact that some proteases do not renature and hence cannot be detected following treatment with SDS. However, it has an advantage the fact that it allows estimation of the relative molecular weight of proteases. Protein separation using nondissociating PAGE is performed using much gentler protease inactivation conditions than those produced by treatment with SDS. Like SDS-PAGE, nondissociating PAGE permits detection of multiple forms of enzymes; however, a disadvantage is that it cannot be used to obtain molecular weight estimates of proteases. The main variable to control during development of zymograms is the length of time of incubations. Increasing incubation (development) time generally increases the sensitivity of protease detection; however, as the length of time of incubation increases so does the extent of diffusion of proteases and substrates. If incubations are prolonged, protease bands will diffuse, decreasing resolution. Additionally, zones of lysis produced by closely migrating proteolytically active species will merge, eliminating the possibility of detecting all proteolytic species in the sample. Zymographic techniques can be extremely useful in identification and characterization of microbial proteases. If a few properties of a protease are known, such as the pH range over which the enzyme is active, and whether it can renature after exposure to SDS, zymographic techniques can be specifically and readily adapted to optimize conditions for detection and assist in characterization of the enzyme.