Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay.
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.
Beekman
B
B
Gaubius Laboratory, TNO Prevention and Health, Department of Vascular and Connective Tissue Research, Leiden, The Netherlands.
van El
B
B
Drijfhout
J W
JW
Ronday
H K
HK
TeKoppele
J M
JM
D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D001681:Biological Assay; D015415:Biomarkers; D005453:Fluorescence; D006801:Humans; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D013582:Synovial Fluid / Q000378:metabolism*
D015415:Biomarkers; D019278:Matrix Metalloproteinase 3