pH, urea and substrate gradients for the optimization of ultrathin polyacrylamide gel zymograms

The preparation of ultrathin polyacrylamide gels with different kinds of gradients (pH, substrates, inhibitors) is described. By using these gels for contact printing after isoelectric focusing with Ampholines or Immobilines and for diffusion tests, the influence of pH or increasing amounts of substrates or inhibitors on enzyme and isoenzyme activities is studied. These methods are successfully applied for the optimization of zymogram techniques and for the easy characterization of industrial microbial enzyme preparations for technological purposes. With buffer-generated pH gradient gels, the pH optimum of all isoenzyme activities is demonstrated by contact printing; the total amount of isoenzyme activities dependent on pH is determined by a diffusion test. Gels with a linear gradient between 0 and 8 M urea are used for isoelectric focusing, diffusion tests and contact printing in order to differentiate the unfolding and denaturing effects of urea on isoenzymes. Alterations in polygalacturonase isoenzyme patterns dependent on urea concentration are not caused by inhibition or denaturation but by the change of charges. In respect to band sharpness and straightness urea can be added advantageously up to 2 M without changing the isoelectric points or activities of the isoenzymes. For the reproducibility of zymograms it is interesting to see that different substrate concentrations reveal different isoenzyme patterns.