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Fluorogenic synthetic substrates are commonly used to monitor the activity of peptidases in vitro. This unit presents a representative protocol that employs (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly~Leu-Dpa-Ala-Arg-NH2) as a substrate to assay matrix metallopeptidases (MMPs). This substrate was first described for the assay of MMP-1, -2 and -3 and it is now widely used as a general MMP substrate. Protocols are given for both stopped-time assays (suitable for assaying MMP activity in a large number of samples) and continuous assays (commonly used when establishing an assay protocol or investigating kinetic aspects of enzyme behavior). Other fluorogenic peptides and protein substrates, together with non-fluorogenic alternatives, are also discussed.

Original publication

DOI

10.1002/0471140864.ps2116s33

Type

Chapter

Publication Date

11/2004

Volume

Chapter 21

Pages

Unit - 21.16

Addresses

Imperial College London, London, United Kingdom.

Keywords

Matrix Metalloproteinases, Peptides, Amino Acid Sequence, Substrate Specificity, Hydrolysis, Kinetics