Equilenin

The ¹H NMR spectrum of KSI Y57F/D40N bound to (9β,13α)-3-hydroxyestra-1,3,5(10)-trien-17-one was acquired at the Stanford Magnetic Resonance Laboratory using an 800-MHz Varian ^UNITYINOVA spectrometer running VNMRJ 3.1A and equipped with a Varian 5-mm triple resonance, pulsed field gradient ¹H[¹³C,¹⁵N] cold probe, as previously described (67 (link)). The sample contained 1 mM KSI and 2 mM equilenin (Steraloids, Newport, RI, USA) in 40 mM potassium phosphate (pH 7.2), 1 mM sodium·EDTA, 2 mM DTT, and 10% DMSO-d₆ (v/v) (Cambridge Isotope Laboratories, Tewksbury, MA, USA). DMSO-d₆ served as the deuterium lock solvent and prevented freezing at low temperatures. The spectrum was obtained in a 5-mm Shigemi symmetrical microtube at −3.5°C, following temperature calibration with a 100% methanol standard. The 1331 binomial pulse sequence was used to suppress the water signal with a spectral width of 35 parts per million (ppm) (carrier frequency set on the water resonance) and an excitation maximum between 14 and 18 ppm (68 ). The data were processed using 10-Hz line broadening and baseline correction applied over the peaks of interest. Chemical shifts were referenced internally to the water resonance.

5-androstene-3,17-dione (5-AND), androstenolone, equilenin and estrone were purchased from Steraloids Inc. (USA). ¹⁵N-Labeled NH₄Cl was purchased from Cambridge Isotope Laboratories Inc. (USA). A Superose 12 gel filtration column was purchased from Amersham Bioscience (USA). All chemicals for the buffer solution were purchased from Sigma (USA). All enzymes for DNA manipulation were purchased from Promega (USA). Oligonucleotides were obtained from Genotech Inc. (Korea).