Optimized for both 72-kDa fibroblast gelatinase(MMP 2) and 92-kDa neutrophil gelatinase(MMP 9), this peptide substrate has the fluorescent Trp residue in subsite P’2 and the dinitrophenol (Dnp) quenching group on the N-terminus. The quenching of the Trp fluorescence in the intact substrate is relieved upon hydrolysis of the P1-P’1 bond, giving rise to a continuously recording fluorescence assay. The residues placed in subsites P3-P’1 and P’3 are optimized for MMP, while Arg is placed in P’4 to enhance solubility. The water solubility of this peptide substrate in assay buffer exceeds the Km value for the enzyme reaction, allowing accurate determination of the kinetic parameters. Abs/Em = 280/360 nm.