Abstract: Objective To improve the method of zymography for MMP-2 and MMP-9 detection in plasma of spontaneously hypertensive rats (SHR). Methods The effects of changed incubation time and working solution components, different pH values of developing buffer and plasma freeze / thaw cycles on the activity of MMPs were observed based on gelatin-containing SDS-polyacrylamide gel electrophoresis (SDS-PAGE)-dependent zymography. The relative activity of plasma MMPs was assayed by optimized zymography in Wistar and SHR rats. Results Gel incubation time reduced from 42 h to 17 h did not affect the test results of zymography. Rinsing step was omitted and renaturing buffer only containing 2.5% Triton X-100, NaN3 and NaCl were removed from developing buffer and deionized water was replaced for distilled water in steps after electrophoresis did not affect the test results of zymography. The pH value within 7.2~8.8 of the developing buffer can be used for zymography. Repeated freezing and thawing plasma samples up to 6 times did not affect the test results of zymography. The activities of MMP-2 and MMP-9 detected by optimized zymography were significantly increased in SHR compared with Wistar rats. Conclusions The optimized zymography is simpler and more economical and the experiment results are the same compared with conventional method.

Key words: gelatin zymography, matrix metalloproteinase, hypertension