Continuously recording fluorescent assays optimized for five human matrix metalloproteinases.
Four new fluorogenic heptapeptide substrates have been synthesized with sequences that are optimized for five human matrix metalloproteinases (MMP). All four substrates are similar to one recently reported by Stack and Gray (1989, J. Biol. Chem. 264, 4277-4281) and have 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 on 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 have been optimized for each MMP, while Arg has been placed in P'4 to enhance solubility. Thus, DNP-Pro-Leu-Ala-Leu-Trp-Ala-Arg has been prepared as a substrate for fibroblast collagenase, DNP-Pro-Leu-Ala-Tyr-Trp-Ala-Arg for neutrophil collagenase, DNP-Pro-Tyr-Ala-Tyr-Trp-Met-Arg for neutrophil collagenase, DNP-Pro-Tyr-Ala-Tyr-Trp-Met-Arg for stromelysin, and DNP-Pro-Leu-Gly-Met-Trp-Ser-Arg for both 72-kDa fibroblast gelatinase and 92-kDa neutrophil gelatinase. These substrates have been characterized with respect to their composition, solubility, optical and fluorescence spectra, and hydrolysis by their target MMP. The hydrolysis rates rival or exceed those of either their natural protein substrates or other synthetic peptides. The solubility of each substrate in assay buffer exceeds the KM value for each reaction, allowing accurate determination of the kinetic parameters. These new substrates should greatly facilitate kinetic studies of the MMP.
Netzel-Arnett
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S
Department of Chemistry, Florida State University, Tallahassee 32306.
Mallya
S K
SK
Nagase
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H
Birkedal-Hansen
H
H
Van Wart
H E
HE
D000595:Amino Acid Sequence; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence
D010455:Peptides; D008666:Metalloendopeptidases