Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media.
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 synovial fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu(EDANS)-Ala-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 TNO211 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.
Beekman
B
B
Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands.
Drijfhout
J W
JW
Bloemhoff
W
W
Ronday
H K
HK
Tak
P P
PP
te Koppele
J M
JM
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D001172:Arthritis, Rheumatoid / Q000201:enzymology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000737:chemistry; D004730:Endothelium, Vascular / Q000201:enzymology; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008666:Metalloendopeptidases / Q000032:analysis* / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D010003:Osteoarthritis / Q000201:enzymology; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods* / Q000706:statistics & numerical data; D013582:Synovial Fluid / Q000201:enzymology
D017077:Culture Media, Conditioned; D005456:Fluorescent Dyes; D009842:Oligopeptides; C100761:TNO 211; D008666:Metalloendopeptidases