A Biotin-PEAC₅-maleimide labeling assay to detect electrophiles

摘要

Recently, we established a biotin-PEAC₅-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 ELISA, 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.