Analysis of Gelatinases in Complex Biological Fluids and Tissue Extracts

Abstract

G elatinase B (matrix metalloproteinase-9 [MMP-9]) and gelatinase A (MMP-2) are two closely related members of the MMP family that efficiently degrade denatured collagens or gelatins (Van den Steen et al, 2002). Specific MMPs play a major role in physiological processes, including angiogenesis, wound healing, bone remodeling, and cell migration. Moreover, MMP-9 and MMP-2 are key effector molecules in inflammation, autoimmunity (Opdenakker and Van Damme, 1994), and cancer (Sehgal et al, 1998). Analysis of both enzymes in complex biological samples, especially those with low gelatinase content, therefore is essential. We combined a miniaturized gelatin affinity chromatography with gelatin zymography and Western blot analysis. This strategy allows extremely sensitive and unambiguous detection of gelatinases. Gelatinases are often detected with specific antibodies or by substrate conversion assays. Gelatinase activity assays measure overall gelatinase activity often with the use of labeled gelatins (Paemen et al, 1996). Because these do not discriminate between gelatinase A and B and even other gelatin-degrading enzymes, specificity is low, especially in the analysis of complex biological samples. Introducing affinity prepurification with the use of monoclonal antibodies enhances the specificity (Hanemaaijer et al, 1998). Moreover, only activated enzymes are recognized. ELISA detects specific forms of MMP-9 or MMP-2. Unfortunately, this method does not necessarily differentiate between pro-enzyme and activated forms. Discrimination between different gelatin-degrading enzymes and their respective activation status may be achieved by zymography or Western blot analysis. Gelatin zymography detects picogram levels of MMP-9 (Masure et al, 1991). The sensitivity of Western blot analysis is usually lower and depends on the antibody affinity for MMP-9. In our hands, a combination of two monoclonal antibodies against mouse MMP-9 resulted in a detection limit of 100 pg of MMP-9. Nevertheless, such low detection limits are frequently not attained because the ratio of MMP-9 versus total protein is generally extremely low in crude samples, whereas the total protein load per lane is limited to 25 g so as not to distort the electrophoresis. After protein overloading, gelatinolysis may be eclipsed in zymography. Interference by gelatinolytic activity from other (abundant) enzymes constitutes an additional problem for zymography. For instance, in stomach extracts, pepsin is abundantly present and because this and other proteases also cleave gelatin, it will mask the gelatinases. To improve the ratio of MMP-9 versus total protein and to exclude interfering gelatinolytic activity, we developed a simple strategy for optimal preparation of complex samples, including tissue extracts. We used a miniaturized affinity chromatography purification step, taking advantage of gelatinases’ strong affinity for gelatin (Masure et al, 1991). For rapid and reproducible purification of the samples, we made use of mini-spin columns (Bio-Rad Laboratories, Hercules, California) and gelatin-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). Equilibration buffer was composed of 50 mM of Tris (pH 7.5), 0.5 M of NaCl, 10 mM of CaCl2, 0.01% Tween 20, and 5 mM of o-phenanthroline; washing buffer 1 had a similar composition except that the concentration of Tween 20 was increased to 0.05%. Washing buffer 2 was with omission of NaCl because high salt concentrations hinder the electrophoresis. The o-phenanthroline was added to the samples as a gelatinase inhibitor to prevent the gelatinolytic activity from breaking down the gelatin from the Sepharose beads, which are used for the affinity purification of the enzymes. The binding of gelatinases to gelatin-Sepharose is not disturbed by the presence of o-phenanthroline because this inhibitor acts by the chelation of the catalytic Zn , whereas binding to gelatin is mediated by the three fibronectin type II–like repeats. Elution buffer was at once the electrophoresis loading buffer and was composed of 100 mM of Tris/HCl (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, and 200 g/ml of bromophenol blue as tracking dye. DOI: 10.1097/01.LAB.0000038556.54069.73