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Label‐Free Detection of Protein Tyrosine Phosphatase 1B (PTP1B) by Using a Rationally Designed Förster Resonance Energy Transfer (FRET) Probe
Author(s) -
Durgannavar Trishaladevi,
Kwon Se Jeong,
Ghisaidoobe Amar B. T.,
Rho Kyungmin,
Kim Ju Hwan,
Yoon SunYoung,
Kang Hyo Jin,
Chung Sang J.
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201800529
Subject(s) - förster resonance energy transfer , lysis , phosphatase , fluorescence , protein tyrosine phosphatase , chemistry , alkaline phosphatase , biochemistry , biophysics , substrate (aquarium) , microbiology and biotechnology , enzyme , biology , ecology , physics , quantum mechanics
Abstract A highly selective detection method of native protein tyrosine phosphatase 1B (PTP1B) is described using a target specific probe equipped with 1‐naphthylamine ( λ ex =330 nm, λ em =445 nm). Irradiation of a mixture of PTP1B and Probe 1 with ultraviolet light of 280 nm (corresponding to PTP1B excitation maximum) resulted in significant fluorescence increase at 445 nm, following FRET characteristics. This phenomenon does not occur with other closely related phosphatases or cellular abundant alkaline phosphatase (APP). Probe 1, the most potent and selective probe, was found to competitively inhibit PTP1B ( K i ≈42 n m ), whereas APP inhibition was found to be in the low micromolar range. Furthermore, Probe 1 discriminates between PTP1B and several other phosphatases. Here, we report real‐time label‐free FRET detection of pure PTP1B as well as induced human PTP1B in Escherichia coli cell lysate. In contrast to 6,8‐difluoro‐4‐methylumbelliferyl phosphate (DiFMUP), a representative fluorescence turn‐on PTP substrate, our FRET probe successfully differentiated human cervical carcinoma cell lysate, SiHa, which has a high expression level of PTP1B, from PTP1B‐knockdown SiHa cell lysate (that is, siRNA was used for PTP1B knockdown).