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

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.