New strategy for selective and sensitive assay of cathepsin B using a dityrosine-based material

作者：Kim Chan Jin; Lee Dong Ik; Zhang Dong; Lee Chang Ha; Ahn K Sung^*

来源：Analytical Biochemistry, 2013, 435(2): 166-173.

DOI：10.1016/j.ab.2013.01.002

• Dityrosine
• Fluorescence
• Cysteine proteases
• Cathepsin B
• Selective assay

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 (lambda(ex) = 284-320 nm, lambda(em) = 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 K-M and k(cat)/K-M values were 2.88 mu M and 3.87 x 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.

• 出版日期2013-4-15