«4 S» Polycyclic Aromatic Hydrocarbon Binding Protein: Further Characterization and Kinetic Properties

Abstract

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 °C even in the absence of the ligand. At 65 °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 °C, whereas at 37 °C higher protein concentrations (4 mg/ml) can be reached. Exposure to guanidine-HCl (3 M) and urea (5 M) for 20 min at 4 °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 °C is biphasic and shows a two-step, first-order kinetic pattern. At 37 °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 °C, whereas at 37 °C the reaction is 10 times as fast. The dissociation equilibrium constants at 0 °C and 37 °C are respectively 2.45 × 10⁻⁹ M and 1.09×10⁻⁹ 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.