Identification of crude and purified bacterial proteinases by agar gel diffusion and agar gel electrophoretic procedures

The double diffusion technique, immunoelectrophoresis, the zymogram technique for proteinases and the casein precipitation inhibition test (GPI-test) were employed for identification of specific bacterial proteinases produced by Aeromonas liquefaciens, Aeromonas salmonicida and Pseudomonas aeruginosa. The precipitation lines observed with both homologous and heterologous proteinase-antiproteinase systems in the double diffusion technique and in immunoelectrophoresis, supported in certain respects previous findings regarding the Aeromonas proteinases, but the reactions were not sufficiently specific to give a confirmation of the real relationships between the proteinases. It is concluded that the double diffusion technique and Immunoelectrophoresis are less specific than the GPI-test for the identification of both crude, and purified, proteinase-antiproteinase systems. The zymograms, in combination with the immunoelectrophoretic patterns, could under certain conditions give useful information about the identity of lines representing the proteinase-antiproteinase precipitates in the double diffusion systems. The number of precipitation lines caused by the crude proteinase solutions in Immunoelectrophoresis decreased during storage of the crude antigens, and the solutions could finally behave like the solution of purified antigen.It was shown that the GPI-test was at least 66 to 225 times more sensitive with respect to antigens, and two to three times more sensitive with respect to antisera than the double diffusion technique, for the three systems examined. This is of methodological importance, as high functional activity may be present in a proteinase solution although the structural conditions are unsuitable or the amount of enzyme protein is too small to allow the development of any precipitation lines in the double diffusion technique.