Detection of Functional Matrix Metalloproteinases by Zymography

Abstract

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease [1][2][3][4][5][6] . Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle [7][8][9][10] . This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel 11 .Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

KCa1.1 Potassium Channels Regulate Key Proinflammatory and Invasive Properties of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

“…Zymography-We used gelatin zymography (Invitrogen) to measure production of pro-MMP-2 in serum-free RA-FLS culture supernatants, as described (24). Recombinant human MMP-2 (R&D Systems) was used as a positive control.…”

Substrate-zymography: a still worthwhile method for gelatinases analysis in biological samples

“…Furthermore, the character of proteolytic bands is analysed by incubating the identical zymograms in PMSF, a serine protease inhibitor; or Pefabloc, an irreversible serine protease inhibitor. The immunological detection of lytic bands can be also performed by Western blotting with antibodies against MMP-2, MMP-9 which recognize the pro-forms of both enzymes (Figure 2, panel D) [33][34][35][36].…”

“…To reach this aim, zymography has been demonstrated to be worthwhile technique in providing information on the spatial distribution of activity, an important parameter which cannot be obtained with the currently available high-throughput (reverse) degradomics platforms [36].…”

Enzymatic activity analysis of MMP-2 and 9 collected by swab from lower limb venous ulcers

“…The contrast between these two elements could then be demonstrated graphically, indicating the degree to which the gelatin in the gel had been broken down. 13 …”