TY - JOUR
T1 - New strategy for selective and sensitive assay of cathepsin B using a dityrosine-based material
AU - Kim, Chan Jin
AU - Lee, Dong Ik
AU - Zhang, Dong
AU - Lee, Chang Ha
AU - Ahn, Ik Sung
PY - 2013
Y1 - 2013
N2 - 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 = 284-320 nm, λem = 400-420 nm) 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 2.88 μM and 3.87 × 103 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.
AB - 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 = 284-320 nm, λem = 400-420 nm) 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 2.88 μM and 3.87 × 103 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.
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U2 - 10.1016/j.ab.2013.01.002
DO - 10.1016/j.ab.2013.01.002
M3 - Article
C2 - 23348078
AN - SCOPUS:84874100049
SN - 0003-2697
VL - 435
SP - 166
EP - 173
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -
