Synthesis of 4-Mercuri-17β-estradiol and Study of Its Reaction with Sulfhydryl Compounds and Proteins

Because enzyme-active sites have been identified by covalent bonding of substrate derivatives, we presumed that a similar approach might be used to characterize macromolecular steroid-binding sites. Accordingly, 4-mercuri-17β-estradiol was synthesized and the structure was determined by analysis of nuclear magnetic resonance spectra. Elemental analysis was compatible with monosubstitution of mercury. Under physiological conditions, 4-mercuri-17β-estradiol reacts with cysteine, 2-mercaptoethanol, and reduced glutathione giving a spectral shift with maximal increase in absorbance at 305 mµ. For the reduced glutathione-4-mercuri-17β-estradiol mercaptide, Δe = 3.4 x 103 m-1 cm-1. One mole of egg albumin reacts with 3 moles of 4-mercuri-17β-estradiol, indicating three reactive sulfhydryl groups in the native protein. The reaction follows second order kinetics: k = 13.5 m-1 sec-1. When 4-mercuri-17β-estradiol reacts with glutathione or egg albumin in aqueous solution, 33 µm 17β-estradiol has no effect on the rate of mercaptide formation. Under generally similar conditions, the rates of mercaptide formation with glutamate dehydrogenase and pyruvate kinase (which have allosteric binding sites for 17β-estradiol) are significantly decreased by 33 µm 17β-estradiol. Similar concentrations of cortisol and estriol (studied with pyruvate kinase) have no effect. Further, mercaptide formation between p-chloromercuribenzoate and pyruvate kinase is not inhibited by 33 µm 17β-estradiol. These observations are compatible with a mechanism in which the steroid moiety of 4-mercuri-17β-estradiol delivers mercury to the allosteric binding site for 17β-estradiol. The resulting increase in local concentration favors mercaptide formation with a reactive residue at or near the binding site. Inhibition of mercaptide formation by 17β-estradiol occurs because it can occupy the binding site and thus exclude 4-mercuri-17β-estradiol. These observations indicate that it may be possible to use 4-mercuri-17β-estradiol for identification of amino acid residues at the steroid-binding sites of high affinity present in steroid-converting enzymes and in receptor proteins of target organs.