title: Immunochemical method to detect proteins that undergo selective modification by 1,2-naphthoquinone derived from naphthalene through metabolic activation.
abstract: Naphthalene undergoes biotransformation by a variety of enzymes to yield 1,2-naphthoquinone (1,2-NQ), a reactive metabolite that binds covalently to proteins. Because this covalent modification is thought to account for naphthalene toxicity, a procedure to detect 1,2-NQ bound to macromolecules is required. In this study, we prepared a polyclonal antibody against 1,2-NQ and examined the specificities of the antibody for various aromatic structures and for the regiochemistry of the quinone functionality. Western blot analysis revealed that the antibody prepared against 1,2-NQ recognized the naphthalene moiety with the ortho-dicarbonyl group, but not with the para-dicarbonyl group; in addition, little cross-reactivity of ortho-quinones with different numbers of aromatic rings (n = 1, 3, 4, 5, 6) was seen. Dot blot and Western blot analyses with the polyclonal antibody enabled quantitative determination of the formation of protein-bound 1,2-NQ during the metabolic activation of naphthalene. The present method can be expected to applicable for the identification of the molecular targets of 1,2-NQ derived from naphthalene in cells and tissues.
authors.affiliation: Doctoral Program in Life System Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Miura
authors.forename: Yoshito
authors.initials: Y
authors.lastname: Kumagai
mesh_terms: D000276:Adjuvants, Immunologic; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D001711:Biotransformation; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004785:Environmental Pollutants / Q000378:metabolism* / Q000633:toxicity; D006433:Hemocyanins / Q000276:immunology; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D007120:Immunochemistry; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D009281:Naphthalenes / Q000378:metabolism* / Q000633:toxicity; D009285:Naphthoquinones / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits
chemical_list: D000276:Adjuvants, Immunologic; D000906:Antibodies; D004785:Environmental Pollutants; D007106:Immune Sera; D009281:Naphthalenes; D009285:Naphthoquinones; D011506:Proteins; C031721:naphthalene; C040756:1,2-naphthoquinone; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin