Structural requirements for the action of steroids as quenchers of albumin fluorescence

Abstract

1. 1. Androgens, corticoids, gestagens, estrogens and related steroids are effective quenchers of the intrinsic fluorescence of bovine serum albumin. The quenching effect involves the formation of a steroid albumin complex which formation constant ( K f ) and free energy of formation (Δ G°) can be determined by fluorescence titration. The fluorimetrically determined Δ G° values range from −6.5 to −7.5 kcal/mol. 2. 2. 5α-Androstane and 5α-pregnane are effective quenchers of albumin fluorescence, in accord with the essentially hydrophobic nature of the steroidalbumin interaction. Introduction of hydroxy or oxo groups in 5α-androstane decreases the fluorescence quenching action, but the effect of each group declines when other polar groups are present in the steroid molecule. Similar effects occur with 5α-pregnane except that 20-hydroxy (or oxo) duo-polar derivatives are more effective than the parent hydrocarbon. 3. 3. Comparison of Δ G° values for steroids differing in a single grouping shows that the steroid-albumin interaction is increased by (a) the benzenoid A-ring; (b) sulfate or carboxylate ions in the vicinity of C-3; (c) the 3-oxo group in place of the 3α-hydroxy 1 (with 5β-pregnane derivatives; not with 5α-androstane derivatives); (d) 17β-acetyl or 17β-hydroxy ethyl residues; (e) acetylated or propionated 17β-hydroxy groups; (f) acetylated or methylated hydroxy groups at the C-3 of estrogens; (g) Δ 5 and Δ 6 double bonds; and (h) the 19β-methyl group. The maximal variation of Δ G° determined by affinityenhancing groups is −0.8 kcal/mol. Conversely, the steroid-albumin interaction is decreased by introduction of (i) oxygen atoms at C-3, C-6, C-11, C-16, and C-17; (j) 17α-ethynyl and 17α-acetoxyl residues; (k) benzoylated or hexahydrobenzoylated β-hydroxy groups at C-17; (1) acetylated and benzoylated hydroxy groups at C-3; and the Δ 1 (conjugated) double bond. Oxo groups at C-3, C-6, C-16 and the 16α, 17α-epoxy group are more effective than the corresponding α-hydroxyl in decreasing affinity, while at C-11 and C-17, the α-hydroxyl is more effective than the β-hydroxyl and the oxo group. The effect of substituents is influenced by the whole molecular structure, particularly, by the stereostructure at the A/B juncture, and the presence of an oxo group at C-17. 4. 4. The stereospecific effect of substituents at different positions in the steroid molecule suggests that with non-aromatic, A/B trans (planar) steroids, binding to albumin primarily involves the (α) rear surface of the B-, C- and D-ring, and possibly, the 17β-side chain. With estrogens and A/B cis (dihedral) steroids, the benzenoid A-ring and electron attracting groups at C-3, respectively, may participate in binding.