New strategy for selective and sensitive assay of cathepsin B using a dityrosine-based material.
The increasing number of reports for disease-related proteases has necessitated materials for the fast, sensitive, and specific assessment of protease activities. The purpose of this study was to synthesize and test a dityrosine-based substrate for the selective assay of a specific cysteine cathepsin. DBDY-Gly-INH)2 was synthesized from the conjugation of N,N'-diBoc-dityrosine (DBDY) with two molecules of glycine and isoniazid (INH) for this purpose. The fluorescence of DBDY (λex=284-320nm, λem=400-420nm) disappeared due to the quenching effect of INH. However, the protease-catalyzed hydrolysis resulted in the release of INH and recovered the fluorescence of DBDY. When reacted with 13 proteases, DBDY-Gly-INH)2 was hydrolyzed by the cysteine proteases only. Meeting the growing need to discriminate cysteine cathepsins (e.g., cathepsins B, L, and S found at high levels in various cancers), DBDY-Gly-INH)2 was tested as a substrate for cathepsins B, L, and S. Only cathepsin B catalyzed the hydrolysis reaction among the three cathepsins. The reaction rate followed the Michaelis-Menten kinetics, and the KM and kcat/KM values were 2.88μM and 3.87×10(3)M(-1)s(-1), respectively, which were comparable to those for the materials reported for the selective assay of cathepsin B. Considering the simple preparation of DBDY-(Gly-INH)2, DBDY-(Gly-INH)2 is believed to be valuable for the sensitive and selective assay of cathepsin B activity.
Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, South Korea.
Chan-Jin
CJ
Kim
Dong-Ik
DI
Lee
Dong
D
Zhang
Chang-Ha
CH
Lee
Ik-Sung
IS
Ahn
D002401:Cathepsin B / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004151:Dipeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D007538:Isoniazid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism
C583091:DBDY-(Gly-INH)2; D004151:Dipeptides; D014443:Tyrosine; C007543:dityrosine; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D007538:Isoniazid