Purification of a benzo[a]pyrene binding protein by affinity chromatography and photoaffinity labeling.
Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo[a]pyrene (B[a]P) have been purified from C57B1/6J mouse liver. Following affinity chromatography on aminopyrene-Sepharose, a single polypeptide of 29,000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29,000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30,000 was estimated by gel filtration chromatography of [3H]B[a]P- and [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 +/- 0.66 nM and a maximum number of binding sites of 2.07 +/- 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29,000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1-azidopyrene.
D000345:Affinity Labels / Q000138:chemical synthesis; D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011721:Pyrenes / Q000138:chemical synthesis / Q000494:pharmacology; D014316:Tritium
D000345:Affinity Labels; D002352:Carrier Proteins; D011721:Pyrenes; C032445:benzopyrene carrier protein, mouse; D014316:Tritium; D001564:Benzo(a)pyrene; C028591:azidopyrene