Basic Methods for Determination of Steroid-Protein Interactions

Abstract

The basic requirement of any procedure for determination of protein binding is its capability of distinguishing the bound portion of a ligand from the free, i.e., unbound** fraction. This may be accomplished by actual physical separation of the two species each of which then can be analyzed; such a system is most lucid and leads to least ambiguous results. In many cases, physical separation is not carried out, and the distinction between the bound and unbound form is based on properties specific for one or the other. Examples are methods in which the biological activity of a compound is drastically changed by binding to a protein, as has been discussed for some of the early observations. Another instance is the measurement of surface tension in cases in which the effect of a surface-active compound is abolished by association with a protein. Changes of spectral properties by interaction with proteins are in the same category.

A review of the earlier work on protein-ligand interactions, including a critical evaluation of methods and presentation of binding data has been given by Goldstein (1949). More recent general treatments have been presented by Klotz (1953) as well as by Edsall and Wyman (1958). Methods have been discussed by Hughes and Klotz (1956) and by Rosenberg and Klotz (1960).