Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin.
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 alpha 2-macroglobulin (alpha 2M). Reconstituted MMP-13/alpha 2M 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 alpha 2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW approximately 28,000) was unable to inhibit MMP/alpha 2M enzyme activity, whereas the low-MW inhibitor BB94 (MW approximately 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/alpha 2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by alpha 2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.
Gaubius Laboratory, TNO Prevention and Health, Leiden, The Netherlands.
B
B
Beekman
J W
JW
Drijfhout
H K
HK
Ronday
J M
JM
TeKoppele
D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000097:blood*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D053509:Matrix Metalloproteinase 13; D008666:Metalloendopeptidases / Q000032:analysis / Q000097:blood*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013876:Thiophenes / Q000494:pharmacology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000032:analysis / Q000378:metabolism*
D005456:Fluorescent Dyes; D011480:Protease Inhibitors; D013876:Thiophenes; D019715:Tissue Inhibitor of Metalloproteinase-1; D000511:alpha-Macroglobulins; D010649:Phenylalanine; D003094:Collagen; C080985:batimastat; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D008666:Metalloendopeptidases