Acta Biomedica Scientifica (Apr 2013)
ZYMOGRAPHIC ANALYSIS OF WATER-SOLUBLE PROTEASES OF VIBRIO CHOLERAE O1 AND О139 SEROGROUPS
Abstract
Aim. Zymographic analysis of water-soluble proteases of Vibrio cholerae O1 и О139 serogroups. Materials and methods. Bacteria cultivated on a casein-yeast agar (рН 7,6) at 37 ° with during one twenty-four hours and washed off physiological solution. Bacterial mass (by the concentration of 109 cell./ml) was treatment by sterile solution of urea in an eventual concentration 4,5 М. After day's display and determination of sterility of got lysate insoluble in urea material (cell walls) was deleted by high-speed centrifugation. Supernatant liquid was exposed to the dialysis, released from insoluble in water sediment centrifugation and freeze dried out. Protease activity was determined in diffusion test in 1 % agarose gel, containing 0,5 % gelatin or 0,5% casein. The spectrum of proteases was analysed by a substrate electrophoresis in the blocks of 8% polyacrylamide gel, impregnated in the process of polymerization gelatin or casein (in an eventual concentration 0,1 %), in presence the dodecylsulphate of sodium. Results. After differential centrifugation of ureal lysate of cells and dialysis there are mainly intracellular water-soluble proteases in the investigated extracts. Diffusion tests showed that all preparations of the investigated strains of Vibrio cholerae possessed protease activity of different intensity. Changing of substrate in diffusion test from gelatin to the casein resulted in the general diminishing of the registered areas of hydrolysis, specifying that a casein fissions less actively by comparison to gelatin. Substrate electrophoresis showed that proteins, making the spectrum of water-soluble pro teases of Vibrio cholerae are extracted by urea, possessed molecular mass, varying within from less than 30 кйа to more than 120 кDа. Strains distinctions are marked in quantitative and high-quality description of spectrums of soluble proteases. Dependence of description of type of water-soluble proteases is educed on used substrate. At the estimation of spectrums of proteases clear confirmation is got, that on gels are impregnated by gelatin electrophoretic mobility of proteases is higher, than in gels, containing a casein. Conclusion. Substrate electrophoresis of preparations of cell-free lysates of Vibrio cholerae showed the presence of a few water-soluble proteases, quantitative and high-quality interstrain distinctions, dependence of spectrum of active proteases from of used substrate.
