A new method for the determination of the binding capacity of testosterone-estradiol-binding-globulin in human plasma

A new method for the quantitative determination of the testosterone-estradiol-binding-globulin, TeBG, in human plasma is described. The concentration of the globulin is measured in terms of the specific binding capacity in plasma for 5α-dihydrotestosterone, DHT. The method is based on equilibrium partition in an aqueous two-phase system containing 10% (w/w) dextran (M w = 4 × 10 5), 7% (w/w) poly-(ethylene glycol) (M n = 6 × 10 3), 0.1M KSCN in 0.005 M phosphate buffer. In this two-phase system more than 99% of the total plasma proteins partition into the lower phase and the partition coefficients for DHT and testosterone are 1.66 and 1.73 respectively. The concentration of the bound and unbound DHT or testosterone in equilibrium with the plasma proteins was determined from the concentration of the steroid in the upper phase. The method is rapid and simple and requires only small quantities of plasma. In contrast to those for testosterone, the results for the binding of DHT are not affected by the presence of transcortin and endogenous steroids in plasma. The intrinsic association constant for the binding of DHT and testosterone to TeBG and the apparent association constant for the binding to albumin were determined from Scatchard-type binding plots. The constants for DHT were 2.2 × 10 9 L mol −1 for TeBG and 8.6 × 10 4 L mol −1 for albumin. The affinity of TeBG for DHT was found to be about 2.4 times that for testosterone. The specific binding capacity values obtained, expressed as μg DHT bound/100 ml plasma, were: men, 1.76, women: 3.00; pregnant women, 12.7.