1. Introduction Sex hormone binding globulin (SHBG) is a selective plasma carrier protein, binding both testosterone and estradiol- 17p with high affinity [ 11. Although it is assumed that SHBG may influence both the hormone action and the metabolism of steroids bound to it [2], there is little evidence for its effect on enzymatic transformations of steroids. Such an effect was demonstrated only for the 17-oxidoreduction of several C1s and Cl9 steroids [3,4] and aromatiza- tion of testosterone [ $61; in both instances the presence of SHBG had an inhibitory effect on the reaction rates. Quantitatively, the transfer of glucuronic acid onto various hydroxysteroids is among the most important catabolic reactions of steroid hormones. The enzyme responsible, UDPglucunit seemed. to be of interest to investigate the effect of SHBG, as a model binding protein, on the enzymatic glucuronosi- dation of testosterone in vitro. In this paper, it is shown that SHBG decreases the yields of testosterone glucuronide formed. This effect, to a lower degree was also demonstrated with the non-specific binder, albumin. The effects of specific 242 and non-specific binding proteins differ in their kinetics. 2. Experimental All chemicals were of analytical grade, the solvents were distilled before use. 4 [14C] Testosterone, specific activity 58.2 mCi/mmol (Radiochemical Centre, Amersham, England), was purified by paper chromato- graphy in system A (see below). Non-labelled steroids were purchased from Koch-Light, Colnbrook, England. Uridine diphosphoglucuronic acid, disodium salt (UDPGA) was obtained from Boehringer, Mannheim, Germany. The protein fraction entiched with SHBG was prepared from full term placental serum, by repeated ammonium sulphate precipitations, followed by affinity chromatography on the polyacrylamide based matrix, Enzacryl AA@ (Koch-Light) with covalently attached 5&-androstane-3a, 17&diol 3-hemisuccinate. The release of protein from its binding to the affmant was achieved by elution with buffered testosterone solution and the steroid was then removed by treatment with charcoal, NORIT A. The procedure in detail has been described elsewhere [ 141. The testosterone bind- ing capacity (TeBC) of purified protein fraction was assessed according to Pearlman et al. [ 151. An aliquot corresponding to TeBC of 2 nmol (approx. 8 mg total protein) in 100 ~1 of buffer was used for incuba- tions. Human serum albumin obtained from Fluka AG, Buchs, Switzerland, 20 mg/sample was used as a non- selective protein.