Purification to homogeneity of the major "4S" PAH binding protein from "non responsive" DBA/2N mouse liver by affinity chromatography.
DBA/2N is a genetically non responsive inbred strain of mice in which administration of polycyclic aromatic hydrocarbons (PAHs) does not induce microsomal monooxygenase activity. DBA/2N mouse liver cytosol contains a polycyclic aromatic hydrocarbon-binding protein that sediments, in a sucrose gradient, at 4S ("4S" PAH-BP). Its binding kinetic and physicochemical properties indicate that this protein is practically indistinguishable from the "4S" PAH-BP identified and characterized in liver cytosol of rats and other PAH responsive rodents including C57 B1/6J mice. "4S" PAH-BP was purified to homogeneity from DBA/2N mouse liver by ammonium sulfate fractionation of the cytosol, followed by Sephadex G-200 chromatography and, finally, affinity chromatography using 1-aminopyrene-Sepharose 6B. This procedure yielded about 50 micrograms of protein from 50-60 g of mouse liver, with a recovery of 18%. "4S" PAH-BP as a complex with 3H-(benzo-a-pyrene) was more than 99% pure. A single band was seen on polyacrylamide gel electrophoresis under non denaturing conditions. H-BaP comigrated with the protein band. 3H-BaP bound to the protein was displaced by PAHs with a specificity identical to that obtained using crude cytosol. On electrophoresis in SDS gels, the purified protein migrated as a single protein band with an apparent molecular weight of 40,000.
Institute of General Pathology and Oncology, I Medical School, University of Naples, Italy.
M T
MT
Masucci
C
C
Masi
E
E
Martini
C
C
Maiello
G
G
Papaleo
V
V
Sica
D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008099:Liver / Q000032:analysis*; D051379:Mice; D008811:Mice, Inbred DBA
D002352:Carrier Proteins; D001564:Benzo(a)pyrene