Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media

Abstract

Matrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synodal fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gin-Gly-LeuGIu(EDANS)-AIa-Lys-NH2), containing the MMP cleavable Gly-Leu bond and EDANS/Dabcyl as fluorophore/quencer combination, was synthesized and characterized as an MMP specific substrate. We show that the fluorogenic assay using ] NO211 is sensitive and can detect MMP activity in culture medium from endothelial cells and untreated synovial fluid (SF) from RA and OA patients, and control subjects. MMP activity in SF significantly increased in the order C < OA < RA, thus the frequent use of OA samples as control in studies on RA is debatable.A ey words: Matrix metalloproteinase; Synovial fluid; I luorescence quenching

Exposure of human cartilage tissue to low concentrations of blood for a short period of time leads to prolonged cartilage damage: An in vitro study

“…However, this correlation was not evident in our experiments. The fact that the MMP assay used measures primarily collagenases and gelatinases (24)(25)(26) could explain the absence of a correlation between proteoglycan release and MMP activity. Collagen damage was not determined in this study.…”

“…General MMP activity was determined in the culture supernatants using the internally quenched fluorogenic peptide substrate TNO211-F (Dabcyl-Gaba-Pro-Gln-Gly-LeuCys[Fluorescein]-Ala-Lys-NH 2 ) in the presence or absence of 12.5 mM BB94 (a general MMP inhibitor) (24)(25)(26). The MMP activity in each sample was calculated as the difference in the initial rate of substrate conversion (linear increase in fluorescence over time, expressed as relative fluorescence units [RFUs] per second) between samples with and those without BB94, normalized against the wet weight of the cartilage, with results expressed as RFUs per second per gram cartilage.…”

The Pathophysiology of Endothelial Function in Pregnancy and the Usefulness of Endothelial Markers

“…The MMP-2 and MMP-9 activity in plasma samples was measured by the solid-phase ELISA method 12 with EDANS/DABCYL FRET peptide (AnaSpec, USA). The results were expressed in reference fluorescence units (RFU).…”

<title>Intraoral fiber-optic-based diagnostic for periodontal disease</title>

“…Because other MMP enzymes associated with periodontal disease recognize a different substrate amino acid sequence [9], we can use a similar strategy to add assays for other MMP enzymes, such as gelatinase and stromelysin. Table 1 lists fluorogenic substrate probes that have been evaluated and used to measure the activity of several matrix metalloproteinases [9,13,14,15]. The activity measurements of MMP enzymes are normally acquired from assays preformed in homogeneous aqueous media with the appropriate parameters optimized (e.g., pH, ionic strength, metal ion co-factors, temperature).…”