A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases.
(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) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly fluorescent 7-methoxycoumarin group is efficiently quenched by energy transfer to the 2,4-dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3.4.24.23) cleaves the substrate at the Gly-Leu bond with a 190-fold increase in fluorescence (lambda cx 328 nm, lambda cm 393 nm). In assays of the human matrix metalloproteinases. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 is about 50 to 100 times more sensitive than dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (kcat/Km) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.
Knight
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CG
Strangeways Research Laboratory, Worts Causeway, Cambridge, UK.
Willenbrock
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Murphy
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003374:Coumarins / Q000378:metabolism*; D005109:Extracellular Matrix / Q000201:enzymology*; D006801:Humans; D008666:Metalloendopeptidases / Q000032:analysis*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D003374:Coumarins; D009842:Oligopeptides; C030123:coumarin; D008666:Metalloendopeptidases