Fluorescence polarization assay and SDS-PAGE confirms matrilysin degrades fibronectin and collagen IV whereas gelatinase a degrades collagen IV but not fibronectin

Matrilysin and gelatinase A are hypothesized to have significant roles in u terine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assay s for the proteolysis of fluorescein-labeled full-length substrates, collag en IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of mat rilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes i n fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. T hese studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates . In addition, they are easier to use than previously described, non-fluore scent methods. The results demonstrate that assays using full-length, biolo gical matrix proteins are more sensitive indicators of MMP-specific substra te activity than peptide based assays.