Identifying Steroid Hormone Binding Sites on Human Serum Albumin by 2D NMR

Abstract

Steroid hormones, such as testosterone and estradiol, are often prescribed to the aging population as an attempt to combat symptoms associated with lower hormone levels (i.e. poor sex drive, weight gain, and fatigue). In human serum, these hormones bind reversibly to the sex hormone binding globulin (SHBG) and to a lesser extent to albumin. The specific sites on albumin and whether hormone binding alters fatty acid (FA) binding have not been established, properties which both could affect delivery to cells and the choice of the appropriate dosage of the hormone to give the desired therapeutic effect. To study binding to human serum albumin (HSA), we examined displacement of 13C-methyl-enriched oleic acid (OA) complexed with HSA in 2D NMR spectra that resolve 9 binding sites and quantify their relative affinities for OA. NMR studies were conducted within physiologically relevant concentrations of HSA, at a molar ratio of 4:1 OA:HSA, and with non-physiological levels of hormone. Testosterone and estradiol displaced OA in a low affinity site for FA, which we previously identified as Sudlow's Drug Site 2. Testosterone also partially displaced OA at FA Site 6, a low affinity FA binding site and a secondary site for certain drugs, as identified by NMR and x-ray. Neither hormone displaced FA in the medium affinity site corresponding to Sudlow's Drug Site 1. Our findings show that these steroids can bind to albumin in sites that are not likely to interfere with FA binding under physiological conditions with molar ratios that are less than 4:1 OA:HSA.