New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain.
A series of new substrates for determining the catalytic activity of cysteine proteinases is described. The rate of hydrolysis by papain was monitored by a fluorescence continuous assay based on internal resonance energy transfer using 5-[(2-aminoethyl)amino]naphtalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) as fluorescent donor and quenching acceptor, respectively, in peptides with the general structure: DABCYL-Lys-Phe-Gly-Xxx-Ala-Ala-EDANS. The substrates were used to evaluate the effect of amino acid structure in the S1' position on the kinetic parameters for papain catalyzed hydrolysis.
García-Echeverría
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School of Pharmacy, University of Wisconsin-Madison 53706.
Rich
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DH
D000595:Amino Acid Sequence; D002384:Catalysis; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates / Q000737:chemistry; D010206:Papain / Q000378:metabolism*; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000737:chemistry
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D010206:Papain