title: Immunochemical analysis of acetaminophen covalent binding to proteins. Partial characterization of the major acetaminophen-binding liver proteins.
abstract: A sensitive immunoassay for detecting acetaminophen (APAP) bound to proteins was developed using an affinity purified antibody directed against the N-acetylated end of the APAP molecule. Western blots of electrophoretically resolved liver proteins taken from mice given an hepatotoxic dose of APAP demonstrated that nearly 85% of the total detectable protein-bound APAP was covalently associated with proteins of 44 and 58 kD. Pretreatment of liver extracts with the sulfhydryl-specific reagent, N-ethylmaleimide (NEM), prior to derivatization with the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), greatly reduced immunochemically detectable APAP-protein adducts and indicated that the antibody detects protein-thiol conjugates of APAP. To investigate the basis of the binding selectivity in vivo, a variety of systems which yielded APAP-protein adducts were analyzed. Systems which activate APAP enzymatically, as in hepatocyte suspensions or in post-mitochondrial (S9) fractions fortified with an NADPH-regenerating system, resulted in a protein binding profile similar to that produced in vivo. Conversely, when extracts or cells were treated with chemically synthesized NAPQI, an alternative protein binding profile was obtained. Two-dimensional electrophoretic analysis of the reduced protein thiol (PSH) content of liver proteins using [3H]NEM labeling revealed that the 58 kD APAP-binding proteins were rich in PSH, whereas the major 44 kD binding protein had virtually no detectable PSH. Many PSH-rich proteins that were not arylated in vivo did bind NAPQI in vitro. However, the 44 kD proteins were not arylated when chemically synthesized NAPQI was added to homogenates or cell suspensions. The present data further suggest that, in addition to the amount and reactivity of free protein sulfhydryls, the cellular localization with respect to the cytochrome P-450 activation site may influence the susceptibility of proteins to NAPQI binding. These findings signal the need for caution in interpreting studies of APAP mechanisms that rely solely on NAPQI addition.
authors.affiliation: Department of Molecular and Cell Biology, University of Connecticut, Storrs 06268.
authors.forename: J B
authors.initials: JB
authors.lastname: Bartolone
authors.forename: R B
authors.initials: RB
authors.lastname: Birge
authors.forename: K
authors.initials: K
authors.lastname: Sparks
authors.forename: S D
authors.initials: SD
authors.lastname: Cohen
authors.forename: E A
authors.initials: EA
authors.lastname: Khairallah
mesh_terms: D000082:Acetaminophen / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000918:Antibody Specificity; D016227:Benzoquinones; D015153:Blotting, Western; D007097:Imines / Q000276:immunology / Q000378:metabolism*; D007158:Immunologic Techniques; D007526:Isoelectric Point; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008970:Molecular Weight; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013438:Sulfhydryl Compounds
chemical_list: D016227:Benzoquinones; D007097:Imines; D011506:Proteins; D013438:Sulfhydryl Compounds; D000082:Acetaminophen; C028473:N-acetyl-4-benzoquinoneimine