"4 S" polycyclic aromatic hydrocarbon binding protein: further characterization and kinetic properties.
A protein that binds polycyclic aromatic hydrocarbons (PAHs) with high affinity and sediments in a sucrose gradient at 4 S has been described in rat liver cytosol. This "4 S" PAH binding protein precipitates at a 40-60% ammonium sulfate saturation. This partial purification procedure allows assay of this protein by using purified 3H-benzo(a)pyrene (3H-BaP) as radioactive ligand and dextran-coated charcoal as adsorbent for unreacted 3H-BaP. The 3H-BaP binding activity measured as a function of pH shows its maximum activity between pH 7.3 and 10.5. The "4 S" PAH binding protein is stable up to 42 degrees C even in the absence of the ligand. At 65 degrees C the binding sites for 3H-BaP are destroyed. The binding activity assayed as a function of protein concentration is linear between 0.4 and 2 mg/ml at 0 degrees C, whereas at 37 degrees C higher protein concentrations (4 mg/ml) can be reached. Exposure to guanidine X HCl (3 M) and urea (5 M) for 20 min at 4 degrees C inhibits the PAH binding completely to the "4 S" protein. Quick dilution or dialysis does not restore the binding activity. The dissociation rate of the "4 S" PAH binding protein measured in the presence of an excess of unlabeled ligand at 0 degrees C is biphasic and shows a two-step, first-order kinetic pattern. At 37 degrees C the dissociation rate is linear and faster, and is complete after 5 min of incubation. The association rate shows the same behavior: the binding is complete after 10 min at 0 degrees C, whereas at 37 degrees C the reaction is 10 times as fast. The dissociation equilibrium constants at 0 degrees C and 37 degrees C are respectively 2.45 X 10(-9) M and 1.09 X 10(-9) M. The high rates of association and dissociation of BaP to "4 S" PAH binding protein were used to set up an assay to exchange radioactive 3H-BaP with cold BaP.
D000818:Animals; D003600:Cytosol / Q000378:metabolism; D005260:Female; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D011083:Polycyclic Compounds; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D011083:Polycyclic Compounds; D014508:Urea; D019791:Guanidine