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

FEBS Letters

Ronday, H.Karel²  Beekman, Bob²  Bloemhoff, Willem⁴  Drijfhout, Jan Wouter³  te Koppele, Johan M.²  Tak, Paul Peter¹ 

[1] Department of Rheumatology, University Hospital Leiden, PO Box 9600, 2300 RC Leiden, The Netherlands;Gaubius Laboratory, TNO Prevention and Health, PO Box 2215, 2301 CE Leiden, The Netherlands;Department of Immunohaematology and Bloodbank, University Hospital Leiden, PO Box 9600, 2300 RC Leiden, The Netherlands;Department of Organic Chemistry, Gorlaeus Laboratory, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands

关键词: Matrix metalloproteinase;    Synovial fluid;    Fluorescence quenching;    APMA;    4-aminophenylmercuric acetate;    Cha;    cyclohexylalanine;    Cys(Me);    S-methylcysteine;    Dabcyl;    4-(4-methylaminophenylazo)benzoyl;    Dnp;    2;    4-dinitrophenyl;    Dpa;    N-3-(2;    4-dinitrophenyl)-l-2;    3-diaminopropionyl;    EDANS;    5-((2-aminoethyl)amino)naphthalene-1-sulphonic acid;    KIU;    kallikrein inhibitor units;    Mca;    (7-methoxycoumarin-4-yl)acetyl;    MMP;    matrix metalloproteinase;    Nma;    N-methylanthranilic acid;    RP-HPLC;    reversed-phase high-performance liquid chromatography;    RS-47;    112;    [4-(N-hydroxyamino)-2R-isobutyl-3S-(thiopen-2-ylthiomethyl)succinyl]-l-phenylalanine-N-methylamide;    TFA;    trifluoroacetic acid;    TOF-MALDI;    time-of-flight matrix-assisted laser desorption-ionisation;    u-PA;    urokinasetype plasminogen activator;   

DOI  :  10.1016/0014-5793(96)00665-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown