Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer.
Interleukin 1 beta converting enzyme (ICE) is responsible for processing an inactive 31-kDa precursor to the active, mature 17-kDa Il-1 beta with cleavage occurring between the Asp116-Ala117 amide bond. We have prepared a peptide substrate that contains the protease cleavage site situated between two fluorophores located at the termini of the molecule. Upon cleavage of DABCYL-Tyr-Val-Ala-Asp-Ala-Pro-Val-EDANS (DABCYL-ICE-EDANS), an increase in fluorescence is observed at the EDANS emission wavelength of 490 nm, permitting a continuous assay of ICE that is useful in the screening of inhibitory compounds. The Km and kcat results for hydrolysis of DABCYL-ICE-EDANS by ICE were 11.4 +/- 1.6 microM and 0.79 +/- 0.4 s-1. The second order rate constant for hydrolysis of this substrate (kcat/Km = 7.0 +/- 1.3 x 10(4) M-1 s-1) is comparable to that for the cleavage of the previously described fluorogenic substrate, Ac-Tyr-Val-Ala-Asp-AMC (6.4 x 10(4) M-1 s-1).
Pennington
M W
MW
Department of Peptide Chemistry, Bachem Bioscience Inc., King of Prussia, PA 19406.
Thornberry
N A
NA
D000595:Amino Acid Sequence; D020170:Caspase 1; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009842:Oligopeptides; C087912:4-(4'-dimethylaminophenylazo)benzoyl-tyrosyl-valyl-alanyl-aspartyl-alanyl-prolyl-valyl-5((2-aminoethyl)amino)naphthalene-1-sulfonic acid; D020170:Caspase 1; D008666:Metalloendopeptidases