Steroid-Albumin Conjugate Interaction with Steroid-Binding Proteins*

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Abstract
A series of progesterone (P)-bovine serum albumin (BSA) conjugates were synthesized by precisely controlling the molar ratios in the reaction mixtures of hemisuccinyloxyprogesterone, isobutyl chloroformate, and BSA. P was conjugated to BSA via the 2α, 11α, or 21 positions. 17β-Estradiol (E2) was conjugated through a 6-carboxymethyloxime side chain to BSA. An additional series of steroid-BSA conjugates was prepared in which fluorescein was attached to the BSA portion. All of the conjugates were tested for binding to rabbit uterine cytoplasmic P and estrogen receptors using charcoal adsorption and sucrose density gradient techniques. P-BSA and E2-BSA conjugates compete against [3H]P and [3H]E2 for the appropriate receptor proteins, but hemisuccinyloxyprogesterones and 6-carboxymethyloximino- E2 do not compete against steroid hormones for the appropriate steroid receptors. 21-P-BSA in the P-BSA series shows the highest degree of competition against [3H]P for P receptor. The maximum competitive activity of 21-P-BSA was observed for a steroid to BSA ratio of 6:1. Substrate activity of P-BSA conjugates with the enzyme 20β-hydroxysteroid dehydrogenase (E.C. 1.1.1.53) showed that activity is maximal with P linked to BSA via the 21 position, and it varies with the steroid to BSA ratio. The highest substrate activity for the 21-P-BSA series is obtained with a P to BSA ratio of 6:1. The presence of a nonsteroid molecule on BSA does not influence the interaction of the steroid moiety of the conjugate with receptor protein. This is shown by competition studies with 21-P-BSA-fluorescein or E2-BSA-fluorescein conjugates with P or estrogen receptor. To be certain that binding activity is solely due to the conjugates and not to any steroid released from the BSA by hydrolysis of a steroid ester linkage, all steroid-BSA conjugates were treated with charcoal before their use in the assays. Steroid covalently linked to BSA retains the ability to interact with receptor and enzyme proteins. Steroid-BSA-fluorescein conjugates have potential for detecting steroid target tissues and cells. (Endocrinology106: 356, 1980)