A new method for measuring covalent binding of chemicals to cellular macromolecules.

摘要: Abstract A new method has been developed for measuring the total covalent binding of metabolically activated compounds to cellular macromolecules. This employs equilibrium dialysis, in presence detergent sodium dodecyl sulfate (SDS), remove unbound radiolabeled compound and its metabolites from [ 14 C] Bromobenzene (80 μM), C]aflatoxin B 1 (5 μM) or 3-[ C]methylcholanthrene (100 was incubated (37°C) with primary hepatocytes liver microsomes isolated Fischer-344 rats. The C-radiolabel hepatic microsomal macromolecules measured by SDS-equilibrium dialysis compared that exhaustive extraction. After h incubation microsomes, 2–7 times more detected than radioactivity associated these migrated discrete positions on SDS-polyacrylamide disc gels. non-dialysable incubations bromobenzene could not be extracted diethyl ether even after treatment dialysin β-glucuronidase-sulfatase dilute acid. taken indicate did include free metabolites, a conclusion supported thin-layer chromatography analysis dialysin. lower amount extraction may related inability trichloroacetic acid quantitatively precipitate small molecular weight is an easy, rapid non-destructive technique binding. macromolecular integrity sample maintained allows further studies concerning specificity interactions.