1. The percentage binding of testosterone (T) and oestradiol (E2) to sex hormone binding globulin (SHBG) and human serum albumin (HSA) was determined over a range of SHBG concentrations of 16–250 nmol of dihydrotestosterone (DHT) bound/l. It was found that the binding of both T and E2 to HSA was a function of their binding to SHBG and bore an inverse relationship to it. After removal of both SHBG and HSA from plasma by affinity chromatography a ‘residual’ binding of about 11% for T and 12% for E2 was still apparent. in addition to the specific high-affinity, low capacity binding of E2 to SHBG, non-specific low-affinity binding of 7–12% was demonstrated after selective denaturation of the specific binding site of the latter.
2. Competition studies indicated that although at the relatively higher levels of SHBG found in the normal female the physiological concentrations of E2, T and DHT need not be taken into account in estimating the unbound fractions of steroids, at the relatively lower levels of SHBG found in normal men and hirsute women, the physiological concentrations of T and DHT are effective in causing statistically significant displacement of E2 from the common, specific binding site on SHBG.
3. A simple computerized technique is described for the determination of fractions of E2 and T respectively, that are unbound to SHBG, unbound to SHBG and HSA, and unbound to all plasma proteins, when the total plasma levels of E2, T, DHT and SHBG are known.