New strategy for selective and sensitive assay of cathepsin B using a dityrosine-based material.
Anal Biochem
; 435(2): 166-73, 2013 Apr 15.
Article
en En
| MEDLINE
| ID: mdl-23348078
ABSTRACT
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.
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Espectrometría de Fluorescencia
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Tirosina
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Catepsina B
/
Dipéptidos
/
Isoniazida
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Anal Biochem
Año:
2013
Tipo del documento:
Article
País de afiliación:
Corea del Sur