Preferential binding of testosterone over epitestosterone by human plasma

The main purpose of this study was to investigate if the specificity of the binding of testosterone to plasma proteins could be defined as a preferential binding of this steroid over epitestosterone. The amount of testosterone that is specifically bound was calculated using the formula: (Formula presented) T concentration, where “R, ” is the ratio (HC) testosterone: (3H) epitestosterone in plasma prior to centrifugation, "RH" is the isotope ratio in the protein-free supernatant obtained after ultracentrifugation (149, 000 x g, at 0 C, for 18 h) and “T concentration” is the testosterone concentration in plasma resulting from addition of (¹⁴C) testosterone, the endogenous steroids having been removed by preliminary charcoal extraction. The theoretical separation of the binding sites for testosterone into two populations, one nonspecific with no preference for testosterone over epitestosterone, and another with absolute specificity for testosterone over its physiologically inactive stereoisomer, proved to be useful. Ovalbumin was found to be an example for non-specific, nonpreferential binding. Determination of the ratio (¹⁴C) testosterone: (³H) epitestosterone in the successive fractions of various ultracentrifuged preparations showed a small but significant preference for (HC) testosterone by human and bovine serum albumin, while ai-acid glycoprotein had a preference for (³H) epitestosterone. Saturation curves showed at least two components: the first one, presumably corresponding to TeBG, had a higher affinity and lower capacity. This binding capacity can be accurately determined by extrapolation to the ordinate of the second component, a straight line corresponding to a binding of somewhat lower affinity and much larger capacity.