D 10 camphorsulfonic acid

Ultra-grade HEPES (N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)) and H₃BO₃ were from Sigma-Aldrich Co. (St. Louis, CA, USA) and Fluka (Munich, Germany), respectively. Biochemical grade Tris (tris(hydroxymethyl)aminomethane) and ultra-grade TCA (trichloroacetic acid) were purchased from Panreac AppliChem (Darmstadt, Germany). Calcium chloride standard solution was from Fluka (Munich, Germany). Strontium chloride standard solution, ultra-grade KOH and EDTA (ethylenediaminetetraacetic acid) acid were from Sigma–Aldrich Co. (St. Louis, CA, USA). Standard solutions of EDTA potassium salt was prepared as described in [20 (link)]. TOYOPEARL SuperQ-650M was purchased from Tosoh Bioscience (Griesheim, Germany). HiPrep 26/60 Sephacryl S-100 HR was from GE Healthcare (Chicago, IL, USA). Sephadex G-25 was a product of Pharmacia LKB (Uppsala, Sweden). A molecular mass marker for SDS-PAGE was a product of Thermo Scientific (Waltham, MA, USA). d-10-camphorsulfonic acid was from JASCO, Inc. (Tokyo, Japan). Other chemicals were reagent grade or higher. All buffers and other solutions were prepared using ultrapure water (Millipore Simplicity 185 system). Plastics or quartz ware were used instead of glassware, to avoid contamination of protein samples with Ca²⁺. SnakeSkin dialysis tubing (3.5 kDa MWCO) from Thermo Scientific (Waltham, MA, USA) was used.

Far-UV circular dichroism (CD) studies were carried out at 25°C using a J-810 spectropolarimeter (JASCO) equipped with a Peltier-controlled cell holder. The instrument was calibrated with an aqueous solution of d-10-camphorsulfonic acid (JASCO) according to the manufacturer’s instruction. The cell compartment was purged with nitrogen. The quartz cells with pathlength of 1.0 mm were used; protein concentration was 11–12 μM. The contribution of buffer (5 mM Na₂HPO₄, 0.88 mM KH₂PO₄, 150 mM NaCl, 1.35 mM KCl, pH 7.0) was subtracted from experimental spectra. Spectral bandwidth was 1 nm, averaging time 2 s, and accumulation 3. The contents of α-helices and β-sheets were estimated using CDPro software package [55 (link)] (http://lamar.colostate.edu/~sreeram/CDPro/main.html).