A Biotin-PEAC₅-maleimide labeling assay to detect electrophiles

Abstract

-Recently, we established a biotin-PEAC 5 -maleimide (BPM)-labeling assay, which can be used to determine the modification of electrophilic metals to proteins (Toyama et al., J. Toxicol. Sci., 38, 477-484, 2013). In the present study, we applied a BPM-labeling assay to detect protein S-modification by environmental organic electrophiles. After exposing A431 cells to 1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ), there was an inverse correlation between Western blot analysis with specific antibody against these electrophiles and that with BPM on the blot intensity to detect protein modification. Similar results were also observed using enzyme-linked immunosorbent assay (ELISA) with BPM. Modification of proteins in mouse liver cytosol by 5-hydroxy-1,4-NQ, 5,8-dihydroxy-1,4-NQ, 1,4-benzoquinone (1,4-BQ), tert-butyl-1,4-BQ, and N-acetyl-p-benzoquinoneimine, an electrophilic metabolite of acetaminophen, was detected using ELISA, but not non-electrophilic quinones or hydroquinone. We also tested whether ELISA could be used to detect electrophiles contained in the vapor phase of ambient air samples collected in the midtown area of Los Angeles. Taken together, the results suggested that the ELI-SA, developed in this study, can detect the existence of electrophilic quinones that covalently modify cellular proteins, resulting in modulation of redox-signal transduction pathways or cell damage.

Polysulfide Na2S4 regulates the activation of PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4-naphthoquinone through formation of sulfur adducts

“…BPM-labeling assay was performed as described previously 40, 41 . Briefly, primary mouse hepatocytes were exposed to 1,4-NQ or 1,4-NQ–S–1,4-NQ-OH, at molar equivalent concentrations of 1,4-NQ (40 µM), for 1 h, then lysates were prepared using RIPA buffer, as described under lysate preparation.…”

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells

“…The BPM precipitation assay was performed according to procedures described previously, with a minor modification (Abiko et al, 2015;Toyama et al, 2013). A homogenate of liver from C57BL/6J male mice was centrifuged at 600 × g for 10 min, the supernatant was centrifuged at 9000 × g for 20 min, and then the lysate obtained was stored frozen under liquid nitrogen and kept at -80°C before use.…”

“…As shown in Fig. 1, 2-hydroxy-1,4-NQ (ω = 6.15 eV), 2,3,5,6-tetrametyl-1,4-BQ (ω = 6.36 eV), 2-methyl-1,4-NQ (ω = 6.39 eV), and 2-hydroxy-3-(3-methyl-2-butenyl)-1-,4-NQ (ω = 6.52 eV) had negligible chemical modification capacities as evaluated by the BPM assay (Toyama et al, 2013;Abiko et al, 2015), whereas other quinones arylated proteins in 9000 × g supernatant of mouse liver (Fig. 1).…”

<i>S</i>-Mercuration of ubiquitin carboxyl-terminal hydrolase L1 through Cys152 by methylmercury causes inhibition of its catalytic activity and reduction of monoubiquitin levels in SH-SY5Y cells

“…To detect the S-modification of recombinant UCH-L1, the BPM labeling assay was performed as described previously Abiko et al, 2015). Briefly UCH-L1 (0.3 μg) was reacted with MeHg in 20 mM Tris-HCl (pH 7.5) at 37°C for 30 min.…”

“…We have developed previously an assay to detect chemical modifications using biotin-PEAC 5 -maleimide (BPM), which can label free thiols Abiko et al, 2015). However, global analysis for detection of S-mercurated proteins is challenging to perform with these assays.…”