Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay

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Abstract
Stromelysin‐1 (MMP‐3) is an important member of the matrix metalloproteinase family. In joint‐degrading diseases like arthritis, elevated levels of MMP‐3 protein are detected in synovial fluid using immunological methods. However, these methods do not discriminate between active and inactive enzyme. In the present study, a specific stromelysin activity assay was developed using the selective fluorogenic substrate TNO003 (Dabcyl‐Gaba‐Arg‐Pro‐Lys‐Pro‐Val‐Glu♦Nva‐Trp‐Arg‐Glu(EDANS)‐Ala‐Lys‐NH2, ♦=cleavage site). For its use in biological media, cleavage of TNO003 by enzymes other than stromelysin was effectively blocked by a proteinase inhibitor cocktail. Spiking of MMP‐3 to synovial fluid resulted in an MMP‐3 concentration‐dependent linear increase in activity. The measured MMP‐3 activity was not affected by the addition of MMP‐13, even in a 5‐fold excess over MMP‐3. Synovial fluid from rheumatoid arthritis patients demonstrated 100‐fold higher levels of active stromelysin than control synovial fluids.