MMP-2 and MMP-9 in Human Peripheral Blood: Optimizing Gelatinase Calibrator for Degradome Research and Discovering a Novel Gelatinolytic Enzyme

Abstract

Matrix metalloprotease-2 and -9 (gelatinase A and B, respectively) are enzymes crucially involved in a plethora of physiopathological conditions. Gelatin zymography is considered one of the major qualitative/semiquantitative assays for simultaneously determining zymogenic, active, and complexed forms of gelatinases. Critical steps are represented by variations in sample collection methods, molecular weight standard calibrators, and different zymography assay protocols. A normalization of these aspects is required for reducing discrepancies in technical procedures and interpreting results among different laboratories. In this study, we describe a novel protocol for gelatin zymography with increased pore size, which improves the separation of gelatinases with different molecular weights. A new method for obtaining gelatinase calibrator for gelatin zymography, by extracting MMP-2 and MMP-9 from peripheral blood, is also reported. Our method provides a gelatinase calibrator with enhanced stability both at room temperature and during multiple freeze−thaw cycles. This calibrator preparation is also suitable for in vitro post-translational modifications. For the first time, the improved zymography protocol allowed us to reveal in human peripheral blood samples new gelatinolytic bands resolved at very high molecular weight, likely complexes of MMP-9, undetectable with classical zymography protocols.

Characteristics of the Intestine Extracts and Their Effect on the Crude Collagen Fibers of the Body Wall from Sea Cucumber Apostichopus japonicus

“…These findings are similar to our results. In addition, gelatinases A and B, namely matrix metalloproteinases 2 and 9, were reported to be synthesized and secreted in vivo as latent pro-enzymes with molecular weights between 72-225 kDa [23]. Therefore, there might also be some precursor enzymes in the extracts of sea cucumber intestines in this study.…”

“…Weak bands were observed at around 133 kDa. This may be due to polymers or precursors of some enzymes [23].…”

“…Weak bands were observed at around 133 kDa. This may be due to polymers or precursors of some enzymes [23]. In order to further clarify the properties of the enzymes in the sea cucumber intestinal extract, a variety of protease inhibitors were used, including 1,10-phenanthroline, E-64 and PMSF.…”

A novel antibacterial biomaterial mesh coated by chitosan and tigecycline for pelvic floor repair and its biological performance

“…In order to further study the possible process of collagen degradation in membranes, MMP9 was detected by immunohistochemistry. MMP9 belongs to the matrix metalloproteinases family and is a kind of gelatinase [ 35 , 36 ]. Our study found that the expression of MMP9 in our antimicrobial membrane was high, which indicated that the collagen fiber was degraded and the biocompatibility was good.…”

Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica

“…The lysed supernatants were quantified using Bradford method and 16.6 μg of total proteins were loaded in each lane. Gelatin zymography was carried out with fixed-concentration (7.5%) polyacrylamide separating gels copolymerized with 3 g/L 90 Bloom Type A gelatin and 4% stacking gel as described previously [ 27 ]. Briefly, zymogram gels were run in cold SDS running buffer (25 mM Tris, 192 mM glycine, and 0.1% w/v SDS) at a constant voltage of 120 V, for about 2 h. After electrophoresis, gels were washed twice for 20 min at room temperature in Triton X-100 2.5% to gently remove SDS.…”