Development of polyclonal antibodies for detection of protein modification by 1,2-naphthoquinone

Naphthoquinones have been reported to be toxic to liver cells in vitro. Protein modification is associated with naphthoquinone-induced cytotoxicity. In addition, 1,2-naphthoquinone was found to bind covalently to cysteine residues of proteins of lung Clara cells incubated with naphthalene. To further identify the target proteins of the naphthoquinone, we raised polyclonal antibodies by immunizing rabbits with 1,2-naphthoquinone protein adducts. A high titer of polyclonal antibodies was obtained by antiserum dilution tests. Competitive ELISA showed that the antibodies specifically recognize the 1,2-naphthoquinone N-acetylcysteine adduct. Very weak cross reactivity toward N-acetylcysteine and its 1,4-naphthoquinone as well as naphthalene oxide adducts was observed. For covalent binding studies, we incubated mouse liver homogenates with 1,2-naphthoquinone at concentrations of 1.0 and 10 microM at 37 degrees C for 1 h. The resulting protein samples were developed by SDS-PAGE, followed by Western blotting and immunostaining using the polyclonal antibodies. Chemiluminescent bands developed with ECL chemiluminescence kit were observed on the poly(vinylidene difluoride) microporous membrane blotted with the mouse liver homogenates exposed to 1.0 and 10 microM 1,2-naphthoquinone. One chemiluminescent band at a molecular weight of 22 kDa was observed in the lane loaded with the protein sample incubated with 1.0 microM 1,2-naphthoquinone, and many chemiluminescent bands at a wide range of molecular weights were observed in the lane loaded with the protein sample incubated with 10 microM quinone. As expected, no chemiluminescent bands were detected on the membrane blotted with the proteins exposed to vehicle. We have successfully raised polyclonal antibodies to recognize 1,2-naphthoquinone cysteine adducts and developed immunostaining to detect protein modification by 1,2-naphthoquinone.