18] Physical-chemical analysis of steroid hormone receptors

Abstract

Definitive evaluation of the mass, chemical composition, and conformation of steroid hormone receptors and of the changes that may occur on interaction with the steroid, the nucleus, or chromatin components will require milligrams of the purified macromolecules. The objective of this chapter is to estimate the physical parameters from nanogram amounts of impure receptors. Density gradient centrifugation—one method of analysis—is the subject of several sections of the chapter and a large proportion of the receptor literature. The chapter deals with three other techniques—gel filtration, gel electrophoresis and isoelectric focusing, and the integration of data from various procedures into a coherent description of the receptor molecule. The limited availability and unique properties of steroid receptors impose certain restrictions on the application of these techniques. The extremely low concentrations of receptors require highly sensitive methods of detection. Comparisons with standard proteins are essential in quantitative gel filtration and electrophoresis. The steroid-binding unit may interact with various contaminants in crude preparations, depending on experimental conditions. An estimate of a physical–chemical parameter is meaningful only if accompanied by a statement describing the precision of that estimate. The standard deviation of a set of measurements is one method of expressing that precision. Confidence limits provide a way of stating both how close a determination is likely to be to the mean value and the chance of its being that close. Analysis of receptors by gel filtration, gel electrophoresis, or any other method necessarily involves a consideration of the statistical factors that determine the uncertainty of the result.