Fatty acid free hsa

Fatty acid-free HSA was purchased from Sigma Aldrich (Milwaukee, USA) and used without further purification. BPB (purity > 99%), BPE (purity > 99%), deuterium oxide (D₂O, 99.9% purity), dimethyl sulfoxide-d6 (DMSO-d6), and 5-dimethylaminonaphthalene-1-sulfonamide (DNSA) were purchased from J&K Scientific, Ltd. (Beijing, China). Dansylsarcosine (DNSS) was obtained from Heowns Biochemical Technology Co., Ltd. (Tianjin, China). All other reagents were of analytical grade and used without further purification.

Fatty acid free HSA at 99% purity (Sigma–Aldrich) and lyophilized ESA with the purity no less than 96% (Equitech-Bio) were additionally purified in a two-step procedure [31 (link)]. The complexes HSA–Myr-2S-cPA and ESA–Myr-2S-cPA were formed by incubation of the respective albumins with a 10-molar excess of Myr-2S-cPA, with constant mixing overnight at room temperature. Prior to incubation, the mixtures were heated to 50°C for approximately 1 h. Both samples were centrifuged before the crystallization setup. The crystallization conditions previously established for HSA [24 (link)] and ESA [31 (link)] were optimized to get crystals of their complexes with Myr-2S-cPA. The best crystals were grown by the hanging drop vapour diffusion method against the solutions containing: 28% PEG3350, 50 mM phosphate buffer at pH 7.5 for HSA–Myr-2S-cPA, or 1.8 M ammonium sulfate, 0.1 M acetate buffer at pH 4.5 for ESA–Myr-2S-cPA. The concentration of albumin used for crystallization was 2 mM for HSA and 1.2 mM for ESA.

Quercetin (purity ≥ 99%) and fatty acid-free HSA (purity ≥ 96%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and indomethacin (purity ≥ 99%) was purchased from Acros Organics, (Geel, Belgium). All chemicals were used without further purification. Binding of Quercetin and indomethacin to HSA was studied using spectrofluorimetry, molecular docking, and molecular dynamics simulations.

Fatty acid free HSA and warfarin sodium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Flavonoids were obtained from Sigma-Aldrich (quercetin), TransMIT GmbH (Gießen, Germany) (isoquercitrin, rutin, hyperoside, cynaroside, and isoorientin), and Extrasynthèse (Genay, France) (luteolin and quercetin-3-O-glucuronide). All standards had a specified purity of ≥98% and have been used without further purification.

GDC-0941 (≥ 99%) was purchased from Selleck Chemicals LLC (Houston, TX, USA; http://www.selleckchem.com; accessed on 5 February 2022). Fatty-acid-free HSA was obtained from Sigma–Aldrich Corporation (Milwaukee, WI, USA). WF-Na and IB were purchased from J&K Scientific Ltd. (Beijing, China). All other analytical-grade reagents were used without purification. The solutions of HSA (20 μM), GDC-0941 (2 mM), phosphate-buffered saline (10 mM, pH 7.4), WF-Na, and IB (2 mM) were prepared before the experiment. Milli-Q water was used in all experiments. The stock solution of HSA was stored at 277 K.

Chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) or BioRad Laboratories (Hercules, CA, USA). 9-Nitrooleate and 10-nitrooleate as pure positional isomers were from Cayman Chemical (Ann Arbor, MI, USA); the purity of both was higher than 98%. NO₂-OA as an equimolar mixture of the 9- and 10- positional isomers was provided by Bruce Freeman's lab (University of Pittsburgh) in a purity of >98%; methanolic stock solutions were prepared. Unless stated otherwise, NO₂-OA was used throughout the study as an appropriate mixture of both positional isomers; only the experiment in Fig. S3 (panel B) was performed with pure 9- or 10-nitro isomers. Fatty acid-free HSA was purchased from Sigma Aldrich (no. A3782) at a purity >99%. All solutions were prepared using Milli-Q water (18.2 MΩ cm⁻¹), Millipore, Bedford, MA, USA.