Quantitative Gel Zymography Reappraised for Matrix Metalloproteinases

A reappraisal of quantitative gel zymography technique for matrix metalloproteinases (MMPs) in human plasma has been used for comparison with commercially available enzyme-linked immunosorbent assay (ELISA).

The determination of MMPs is relevant in numerous pathophysiological conditions, as imbalanced MMP activity is associated with many clinical conditions, including cardiovascular diseases, especially if associated with extracellular matrix remodeling, but the results obtained are closely linked to the method used and are not directly comparable.

Scientists at the Italian National Research Council (CNR; Pisa, Italy) obtained heparinized blood samples from 25 volunteers. The samples were centrifuged and the serum stored in aliquots at −80 °C. Gel zymography was performed based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on the use of gelatin 0.5% as substrate for detection of MMP enzymatic activity. Gelatinolytic activities were detected as unstained bands against the background of Coomassie Blue stained gelatin. In order to quantify the concentrations of the analyzed protein in the unknown samples, a dose-response curve was built in each run.

Total MMP-9 was measured in diluted plasma by specific immunometric assay (R&D Systems; Minneapolis, MN, USA), previously standardized. The working range was 0.19 ng/mL to 16 ng/mL, the sensitivity was 0.05 ± 0.01 ng/mL, and the within-assay variability was 414.3 ± 23.5 ng/mL (5.7%), while the between-assay variability was less than 15%. The concentration of total MMP-9 was measured in parallel using the zymography and ELISA for comparison purposes.

The results obtained by gel zymography analysis of human blood samples using different acrylamide/bis acrylamide ratios indicated that 7.5% gels are the more suitable for visualizing MMP-9 forms. In all conditions, four bands with gelatinolytic activity are found: MMP-9 dimers, MMP-9– tissue inhibitor of metalloproteinase1 (TIMP-1) complex, MMP-9, and MMP-2. The time course of the proteolytic activity of MMPs indicated 16 to 24 hours as optimal incubation time and these optimal parameters were used for routine assay. A linear positive correlation was found between the MMP-9 values of zymography and ELISA with the ELISA values significantly lower than those obtained with zymography.

The authors concluded that the main advantage of gel zymography is that it allows visualization of both the latent and active forms of gelatinases. The presence of MMP dimers and MMP/TIMP complexes in the plasma samples, revealed by gel zymography, could thoroughly characterize the role of MMP in associated diseases. Gel zymography allows separation and visualization of MMP-2 bands, so both MMP-2 and MMP-9 can be determined in the same run from a single biological sample. The study was published in the September 2014 issue of the Journal of Clinical Laboratory Analysis.

Related Links:
Italian National Research Council
R&D Systems