ABSTRACT
The present work deals with the determination of association constants at equilibrium by a non-graphical method in binding systems containing one specific receptor. Equations have have been derived from that originally described by Lea (Biochim. Biophys. Acta, 322, 68--74), the terms of which are obtained from the data of simple displacement curves of a bound radioactive ligand by unlabelled competitors identical or different in nature. By knowing the function relating the variations of the bound ligand (B) to the affinity constant (Ki) and the quantity (Mi) of competitor for a given system, it is possible to calculate any of these parameters when the two others are measured. Thus, it becomes easy to compare the relative affinities of differents receptors for the same ligand or that of one receptor for various labelled or unlabelled ligands. Furthermore, theoretical displacement curves can be drawn and compared to experimental data, when only knowing the affinity constant of a specific binding system in given conditions. These modes of calculation have been tested in a study of interactions between various steroids and a fraction of human serum proteins precipitated by ammonium sulfate (30-45%) and containing the sex hormone-binding globulin. Association constants thus obtained agree well with those reported in the literature and determined by graphical procedures.
Subject(s)
Dihydrotestosterone , Ligands , Binding Sites , Kinetics , Mathematics , Methods , TestosteroneABSTRACT
A method for the separation of serum conjugated steroids, sulfates and glucuronides, is described. It is based on the fact that methylene blue forms complexes with ester-sulfates which can be quantitatively and specifically extracted by chloroform from aqueous phase. The quantitative determination of the conjugated fractions thus obtained can be done after solvolysis followed by hydrolysis by beta-glucuronidase from Helix pomatia.
