Biochemical Analysis of Nitrated Matrix Metalloproteinase (MMP) in Synovial Fluid from Patients with TMD

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Nitric oxide (NO), super oxide, and their cytotoxic reaction product peroxynitrite (ONOO) are terminal mediators of cellular injury in various forms of inflammation. Recent studies suggested that these free radicals in the temporomandibular Joint (TMJ) might amplify damage of articular tissues. In the past study, we developed an easy assay system using a erogenic peptide for measuring matrix metalloproteinase (MMP) activity in synovial fluid (SF) and found that MMP-3 activatio was a hallmark indicating an early degenerative change in TM disorders (TMD). However, the activating pathway of MMP in vivo remains unexplained. Recently some studies have reported that activates MMPs in vitro. In this study, we examined whether proteins in SF from TMD patient nitro tyrosine, which was known to be stable and specific footprint showing nitration by peroxynitrite. Furthermore, in order to identify nitrated protein in SF, SF proteins were analyzed by immunoprecipitation method. As the results, we demonstrated that nitrated proteins in SF were identified as MMP-2 and -3, and that peroxynitrite in SF activated MMP-3.