A novel coumarin‐labelled peptide for sensitive continuous assays of the matrix metalloproteinases

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Abstract
(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 flourescent 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 (λcm 328 nm, λcm 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 (k cal/K m) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.