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Fluorogenic MMP activity assay for plasma including MMPs complexed to α2-macroglobulin

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Author: Beekman, B. · Drijfhout, J.W. · Ronday, H.K. · TeKoppele, J.M.
Type:article
Date:1999
Institution: Gaubius instituut TNO
Source:Annals of the New York Academy of Sciences, 878, 150-158
Identifier: 235117
doi: doi:10.1111/j.1749-6632.1999.tb07681.x
Keywords: Health · Biomedical Research · Alpha 2 macroglobulin · Batimastat · Fluorescein · Matrix metalloproteinase · Arthritis · Cancer · Collagen degradation · Conference paper · Controlled study · Enzyme active site · Enzyme activity · Enzyme assay · Fluorescence analysis · Human · Molecular weight · Alpha-Macroglobulins · Collagen · Collagenases · Fluorescent Dyes · Humans · Kinetics · Matrix Metalloproteinase 13 · Metalloendopeptidases · Phenylalanine · Protease Inhibitors · Substrate Specificity · Thiophenes · Tissue Inhibitor of Metalloproteinase-1

Abstract

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor α2-macroglobulin (α2M). Reconstituted MMP-13/ α2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by α2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW ~ 28,000) was unable to inhibit MMP/α2M enzyme activity, whereas the low-MW inhibitor BB94 (MW ~ 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/α2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by (α2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.