This work examines the feasibility of using a pH-sensitive fluorescent protein as a molecular reporter for enzyme-catalyzed prodrug activation reaction. Specifically, a ratiometric pHluorins was examined for detection of the activity of horseradish peroxidase (HRP) for the activation of indole-3-acetic acid. The pHluorins and HRP were conjugated chemically, forming a biocatalyst with a self-reporting function. Results showed that the characteristic fluorescence intensity ratio of the conjugate shifted from 1.47 to 1.40 corresponding to the progress of the prodrug activation reaction. The effectiveness of applying the conjugate for inhibition of the growth of Bcap-37 cells was also demonstrated simultaneously with reaction monitoring. The results reveal a very promising approach to realizing in situ monitoring of enzyme activities based on pH shifting for enzyme-based prodrug therapy applications.
Bibliographical noteFunding Information:
This study was supported by the National Natural Science Foundation (31471659, 31300660, and 1303050), the Science and Technology Innovation Action Plan of Shanghai (14431904300), and the Shanghai Pujiang Rrogram (13PJD012) and was partially supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.
© 2016 International Union of Biochemistry and Molecular Biology, Inc.
- enzyme/prodrug therapy
- ratiometric pHluorins