ACCURATE MEASUREMENT OF STEROID-PROTEIN BINDING BY STEADYSTATE GEL FILTRATION

1. (1) A study of gel filtration has been carried out with the object of improving on present methods of observing steroid-protein interaction. 2. (2) Extensive dissociation of the cortisol/corticosteroid-binding-globulin complex, as well as of the cortisol/albumin complex, occurs in gel filtration by current methods. It is shown that this is not avoided by using low temperatures. The dissociation is shown to be highly dependent on the size of the applied sample. This has a major influence on the results of conventional methods of zonal analysis of steroid-protein mixtures by gel filtration. 3. (3) These effects are eliminated when large samples are used to generate steady-state conditions in gel filtration (frontal analysis). A method based on this principle is proposed for the accurate analysis, at 37° in whole serum, of cortisol-protein equilibria. Kinetic factors are rendered unimportant by the method. Protein concentration is unaltered and equilibrium is undisturbed. Column performance is not critical. The cortisol-plasma-protein binding systems are particularly amenable to this method of analysis, because of their high rate of change of equilibrium, and the nature and migration rates of the cortisol-protein complexes. © 1969.