Activity-based enrichment of matrix metalloproteinases using reversible inhibitors as affinity ligands.
Matrix metalloproteinases (MMPs) are zinc dependent metalloproteases characterized by the ability to cleave extracellular matrix and many other extracellular proteins. MMP activity is tightly regulated but disturbances in this regulation can contribute to various disease processes characterized by a progressive destruction of the extracellular matrix. The ability to profile classes of enzymes based on functionally related activities would greatly facilitate research about the involvement of MMPs in physiological and/or pathological states. Here we describe the characterization of an affinity sorbent using an immobilized reversible inhibitor as a stationary phase for the activity-based enrichment of MMPs from biological samples. With a ligand density of 9.8 mM and binding constant of 58 micromol/l towards MMP-12, the capturing power of the affinity sorbent was strong enough to extract MMP-12 spiked into serum with high selectivity from relatively large sample volumes. Experiments with endogenous inhibitors revealed that MMP-12 extraction is strictly activity-dependent, offering powerful means to monitor MMP activities in relation to physiological and/or pathological events by using affinity extraction as a first step in an MMP profiling method.
D000345:Affinity Labels; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D053508:Matrix Metalloproteinase 12; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism*; D011485:Protein Binding
D000345:Affinity Labels; D011480:Protease Inhibitors; D008666:Metalloendopeptidases; C507122:MMP12 protein, human; D053508:Matrix Metalloproteinase 12