Kinetics of the association of several tritiated PCDD and PCDF congeners with the cytosolic Ah receptor from the Sprague-Dawley rat

Abstract

A series of [ 3H]-PCDD and [ 3H]-PCDF congeners of high specific activity were prepared by the chlorination of 1,6- 3H 2]-dibenzo-p-dioxin and 1,4,6-[ 3H 3]-dibenzofuran followed by high pressure liquid chromatographic separation of the resultant mixtures. The compounds synthesized were tritiated 2,3,7,-TrCDD; 2,3,7,8-TCDD; 1,2,3,7,8-PeCDD; 1,2,7,8-TCDF; 2,3,7,8-TCDF; and 1,2,3,7,8-PeCDF. Direct binding studies with male Wistar rat hepatic cytosol showed that the K D values for all six congeners were approximately 5–10 nM. The lack of structure-dependent binding affinities to the Ah receptor is in contrast to previous competitive receptor binding studies and the well-known structure-induction and structure-toxicity relationships for these compounds. The kinetics of binding for each of these ligands to the Ah receptor, and the rate of thermal inactivation of the unliganded receptor were determined at several temperatures. Activation parameters were obtained using both graphical and computational methods; they varied with the identity of the ligand and exhibited substantial experimental uncertainties. The activation parameters were used to computationally determine revised K D's for the congeners which were in the pM range.