Monitoring Metalloproteinase Activity Using Synthetic Fluorogenic Substrates

Fluorogenic synthetic substrates are commonly used to monitor the activity of proteinases in vitro. This unit describes an assay for matrix metalloproteinases (MMPs) using the substrate MCA-Pro-Leu-Gly~Leu-Dpa(Dnp)-Ala-Arg-NH2, where MCA is the fluoro-phore (7-methoxycoumarin-4-yl)acetate, Dnp is the quencher dinitrophenyl, and the tilde (~) represents the peptide-bond target of hydrolysis. This substrate was first described by Knight et al. (1992) for the assay of matrix metalloproteinase (MMP)-1, -2, and -3, and is now widely used as a general MMP substrate (Fig. 21.16.1). Protocols are given for both stopped-time (see Basic Protocol 1) and continuous (see Basic Protocol 2) assays. The former is suitable for assaying MMP activity in a large number of samples, while the latter is commonly used when establishing an assay protocol or investigating kinetic aspects of enzyme behavior. Other fluorogenic peptide and protein substrates are also discussed, along with nonfluorogenic alternatives.

MMP concentration can be estimated from absorbance at 280 nm, but for accurate determination, a method for titrating the amount of proteinase is presented (see Support Protocol).

For a more thorough discussion of MMPs and TIMPs, see unit 21.4. NOTE: Perform all assays at least in duplicate.

Figure 21.16.1 Schematic diagram of MMP hydrolysis of MCA-Pro-Leu-Gly~Leu-Dpa(Dnp)-Ala-Arg-NH2. This MMP substrate contains a methoxycoumarin (MCA) fluorophore and a dinitrophenyl (Dnp) quencher located on opposite sides of the susceptible peptide bond, indicated by a tilde (~). The excitation peak of the quencher overlaps with the emission peak of the fluorophore, allowing the quencher to absorb the energy from the fluorophore in a distance-dependent manner. This prevents fluorescence of the intact substrate by a process of fluorescence resonance energy transfer (FRET). Upon hydrolysis of the susceptible peptide bond, the quencher and the fluorophore become physically separated and the fluorescence of the MCA group can be detected at 393 nm upon excitation at 325 nm.

Contributed by Linda Troeberg and Hideaki Nagase

Current Protocols in Protein Science (2003) 21.16.1-21.16.9 Copyright © 2003 by John Wiley & Sons, Inc.


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