A simple, rapid, and precise method for calculating the steroid hormone-receptor complex concentration in the presence of saturating levels of hormone by using “differential dissociation” techniques
✍ Scribed by Hélène Richard-Foy; Gérard Redeuilh; Robert Richard-Foy
- Publisher
- Elsevier Science
- Year
- 1978
- Tongue
- English
- Weight
- 799 KB
- Volume
- 88
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
The steroid hormone-receptor complex concentrations measured by "differential dissociation" techniques have to be corrected to obtain the true concentrations of receptor binding sites (B,). For the calculation of B,, the parameters kn (product of the equilibrium association constant and the concentration of binding sites of the "nonspecific" component) andf (fraction of the nonspecific binding measured in the experimental estimates of bound ligand by a given technique), previously proposed by Blondeau and Robe1 (J. P. Blondeau and P. Robel, 1975, Eur. J. Biochem. 55, 375-384) are important. A new parameter of interest, E [E = knfi(kn + l)], is discussed. The measurement of this parameter E for three "differential dissociation" techniques allows the comparison of their efficiency and their reliability under various conditions for hormone receptor measurement in cytosol. Charcoal and hydroxylapatite methods are more efficient than the Sephadex G-25 filtration method. It is demonstrated that the "isotopic dilution" correction generally used for the estimation of the background of a given technique may be incorrect whatever the method of correction. A new method, the "double concentration measurement," IS developed. This method is simple, rapid, and precise. It requires two receptor binding measurements at two different saturating concentrations of ligand. This method allows the measurement of the estradiol receptor binding activity from calf uterine cytosol, with an error of less than 5% in samples containing the receptor either free or previously complexed with radioactive hormone, even in the presence of very high concentrations (~0.5 PM) of radioactive steroid.
The precise measurement of the concentration of a steroid hormone receptor is difficult. A "nonspecific" binding is superimposed on the specific binding of the hormone to the receptor. The contribution of this r Preceding papers published under the name of Truong.