title: A dityrosine-based substrate for a protease assay: application for the selective assessment of papain and chymopapain activity.
abstract: N,N'-diBoc-dityrosine (DBDY), which was synthesized by the oxidative C-C coupling of 2 N-Boc-L-tyrosine molecules, was conjugated with two isoniazid (INH) molecules. Due to the quenching effect of INH, DBDY-(INH)(2) lacks the fluorescence of DBDY. As such, it was tested for use in the detection of proteases by measuring fluorescence recovery. In this study, serine proteases (chymotrypsin, trypsin, subtilisin, and proteinase K), metalloproteases (thermolysin and carboxypeptidase A, dispase, and collagenase), aspartic proteases (pepsin and aspergillopepsin) and cysteine proteases (papain and chymopapain) were chosen. Reported optimum assay conditions were chosen for each enzyme. Only papain and chymopapain catalyzed the hydrolysis of DBDY-(INH)(2) and led to fluorescence recovery, possibly due to their extensive binding sites and the INH-mediated inhibition of metalloproteases and aspartic proteases.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, South Korea.
authors.forename: Chan-Jin
authors.initials: CJ
authors.lastname: Kim
authors.forename: Dong-Ik
authors.initials: DI
authors.lastname: Lee
authors.forename: Chang-Ha
authors.initials: CH
authors.lastname: Lee
authors.forename: Ik-Sung
authors.initials: IS
authors.lastname: Ahn
mesh_terms: D055162:Biocatalysis; D002917:Chymopapain / Q000378:metabolism*; D057075:Enzyme Assays; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010206:Papain / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry
chemical_list: D014443:Tyrosine; C007543:dityrosine; D010206:Papain; D002917:Chymopapain