A rapid and sensitive method for determination of covalent binding of benzo[a]pyrene to proteins

A method is presented for the quantitative determination of covalent binding of metabolically activated benzoa]pyrene to microsomal proteins. After incubation of radiolabelled benzoa]pyrene with microsomes and NADPH, the mixture is applied to filter paper discs. These are immersed in ethanol to precipitate the proteins. Unbound radiolabel is removed by repeated washes of the filters in organic solvents before scintillation counting. The method is simple, rapid, sensitive and accurate, and works both with 14C- and 3H-labelled compounds. The method is suitable for measuring the incorporation of other radiolabelled xenobiotics to proteins of both microsomes and other subcellular fractions and for the analysis of binding to isolated proteins.