A new methodology for monitoring the activity of cdMMP-12 anchored and freeze-dried on Au (111).
Matrix metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that are capable of degrading extracellular matrix proteins, but also can process a number of bioactive molecules. They are involved in the cleavage of cell surface receptors, but are also thought to play a major role on cell behaviors as well as in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases, and cancer. For these reasons, it is obvious that a control over MMPs activity is highly desirable. Consequently, the frantic search for new inhibitors has been coupled to the development of high-throughput methods able to rapidly screen the effect of possible MMP inhibitors on the activity of these enzymes. In this scenario, solid-state-based methods play a major role because of their compatibility with array formats that are able to extract more information from smaller sample volumes and offer some important advantages that are not available in the standard solution assays. In this work, the catalytic domain of MMP-12 was immobilized on a gold substrate and the surface coverage was measured by FT-SPR experiments. A new experimental procedure was developed to freeze-dry the anchored molecules and their activity was measured by ESI-MS. The kinetics parameters obtained for the immobilized enzyme are in good accordance with those reported for similar systems in solution. Inhibition of the immobilized molecules was also carried out, demonstrating the applicability of the method for rapid screening of MMP inhibitors.
Grasso
Giuseppe
G
Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici, Bari, Italy.
Fragai
Marco
M
Rizzarelli
Enrico
E
Spoto
Giuseppe
G
Yeo
Kwon Joo
KJ
D020134:Catalytic Domain; D004800:Enzymes, Immobilized / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D005612:Freeze Drying; D006046:Gold / Q000737:chemistry*; D006801:Humans; D053508:Matrix Metalloproteinase 12 / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D004800:Enzymes, Immobilized; D006046:Gold; D053508:Matrix Metalloproteinase 12