title: Determination of matrix metalloproteinase activity using biotinylated gelatin.
abstract: Matrix metalloproteinases (MMPs) and, specifically, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are strongly associated with malignant progression and matrix remodeling. These enzymes are a subject of intensive studies involving screening of comprehensive chemical libraries of synthetic inhibitors. There is no simple method available for measurement of activity of gelatinases and related MMPs. Here, we report a simple, inexpensive, and highly sensitive assay for MMP activity. The assay performed in a 96-well microtiter plate format employs biotin-labeled gelatin (denatured collagen type I) as a substrate. Following the substrate cleavage, only the proteolytic fragments bearing biotin moieties are captured by streptavidin coated on the plastic surface and the captured fragments with at least two biotin molecules should be revealed by streptavidin conjugated with horseradish peroxidase. The frequency of lysine residues is low in collagen type I relative to the MMP cleavage sequences (PXGX). Accordingly, the majority of the cleavage products must be devoid of biotin or possess only one biotin group. Both of these types of fragments cannot be recognized by the horseradish peroxidase-streptavidin conjugate. Therefore, higher gelatinolytic activity is associated with lower signal in the assay. This 2-h assay allows identification of gelatinolytic activity of MMP-2 in concentrations as low as 0.16 ng/ml. The sensitivity of this ELISA-like assay is comparable to that of gelatin zymography, a method widely used to detect gelatinases. However, in contrast to zymography, the assay directly measures the enzymatic activity of MMP samples. The gelatinolytic activity assay permits efficient analyses and screening of the MMP inhibitor panels and allows quantitation of gelatinolytic activity of various MMPs in solution as well as on cell surfaces.
authors.affiliation: Burnham Institute, 10901 North Torrey Pines Road, La Jolla, California, 92037, USA.
authors.forename: B
authors.initials: B
authors.lastname: Ratnikov
authors.forename: E
authors.initials: E
authors.lastname: Deryugina
authors.forename: J
authors.initials: J
authors.lastname: Leng
authors.forename: G
authors.initials: G
authors.lastname: Marchenko
authors.forename: D
authors.initials: D
authors.lastname: Dembrow
authors.forename: A
authors.initials: A
authors.lastname: Strongin
mesh_terms: D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002624:Chemistry, Clinical / Q000379:methods; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005780:Gelatin / Q000378:metabolism / Q000494:pharmacology*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020784:Matrix Metalloproteinase 8 / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D018076:DNA, Complementary; D006877:Hydroxamic Acids; D007211:Indoles; D011480:Protease Inhibitors; D001710:Biotin; D005780:Gelatin; D019809:Streptavidin; D006735:Horseradish Peroxidase; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D020784:Matrix Metalloproteinase 8; C119203:ilomastat; D008239:Lysine