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

[摘要]

(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 (λ_cm 328 nm, λ_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 (k _cal/K _m) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.

[学科分类] 生物化学/生物物理

[关键词] Fluorescence quenching;Endopeptidase;Peptide synthesis;Specificity constant;Dnp;2;4-dinitrophenyl;Dnp-F;1-fluoro-2;4-dinitrobenzene;Dpa;N-3-(2;4-dinitrophenyl)-l-2;3-diaminopropionyl;Fmoc;9-fluorenylmethoxycarbonyl;HOBt;1-hydroxybenzotriazole;Mca;(7-methoxycoumarin-4-yl)acetyl;PyBOP;benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate;TFA;trifluoroacetic acid