Effect of chemical perturbation with sodium thiocyanate on receptor-estradiol interaction. A new exchange assay at low temperature

Abstract

When 0.5 M sodium thiocyanate was added to uterine cytosol previously labeled with excess [3H]-17.beta.-estradiol [E2], no change was detected in the steady-state cytosol concentration of [3H]E2-receptor complex for at least 20 h at 4.degree. C. However, the rate of exchange of bound E2 in the presence of NaSCN was substantially higher than that in the absence of the chaotropic salt. In the presence of NaSCN, the dissociation rate of the complex increased about 10-fold (K-1SCN = 1.10 .times. 10-2 min-1 vs. K-1 = 1.07 .times. 10-3 min-1) while the rate of association increased about 2-fold (K1SCN = 1.2 .times. 107 min-1 M-1 vs. K1 = 7.4 .times. 106 min-1 M-1). The Kd changed 6.4-fold (KdSCN = 9 .times. 10-10 M vs. Kd = 1.4 .times. 10-10 M) with no decrease in the number of binding sites as shown by Scatchard plots of saturation experiments. This effect of NaSCN was exploited to assay preformed estrogen-receptor complex by exchange with [3H]E2 in the presence of 0.5 M NaSCN, there was a quantitative exchange of nonlabeled for labeled estradiol without loss of binding sites. Hormonal steroids other than estrogens did not interfere, and the exchanged estradiol was bound with high affinity. Precision, accuracy and linearity of the method were highly satisfactory.