Trimethylamine n oxide

Ammonium ferrous sulfate hexahydrate ((NH₄)₂Fe(SO₄)₂.6H₂O), horse spleen apoferritin in 0.15 M NaCl, ethanol (C₂H₆O), horse spleen ferritin in 0.15 M NaCl, hydrogen peroxide (H₂O₂), 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO), sodium hydroxide (NaOH), trimethylamine N-oxide (Me₃NO), L-ascorbic acid (C₆H₈O₆), riboflavin (C₁₇H₂₀N₄O₆), ferrozine, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were obtained from SIGMA-Aldrich (Saint-Louis, MO, USA); Coomassie brilliant blue G 250 was obtained from Fluka (Buchs, Switzerland); hydrochloric acid (HCl) from ITES (Vranov nad Toplou, Slovakia); potassium thiocyanate (KSCN) from Slavus (Bratislava, Slovakia); phosphoric acid (H₃PO₄) and ethanol (C₂H₆O) from Centralchem (Bratislava, Slovakia); and distilled water.

Trimethylamine N-oxide (Sigma-Aldrich, Saint Louis, MO, USA) solubilized in culture medium or Tyrode standard solution according to the protocol used, was freshly prepared for each experiment. Menadione (MEN) (Sigma-Aldrich) was solubilized in DMSO at an initial concentration of 100 mM and then diluted in culture medium to the final concentration of 100 μM. ATP was prepared in Tyrode standard solution at a concentration of 10 mM and diluted in culture medium to the final concentration of 100 μM. Tyrode standard solution used in different experiments contained (in mM): 154 NaCl, 4 KCl, 1 MgCl₂, 5.5 D-glucose, 5 HEPES, 2 CaCl₂, pH adjusted to 7.34 with NaOH. Unless otherwise specified, all reagents for cell culture and experiments were purchased from Sigma-Aldrich.

Commercially lyophilized preparation of Ribonuclease-A (type III-A) from bovine pancreas (RNase-A) was purchased from Sigma Chemical Co. Trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, glycine, β-alanine and cytidine 2′–3′ cyclic monophosphate (C>p) were also purchased from Sigma Chemical Co. Guanidinium chloride (GdmCl) and urea were obtained from MP Biomedicals. These and other chemicals, which were of analytical grade, were used without further purification.
RNase-A solution was dialyzed extensively against 0.1 M KCl solution at pH 7.0 and 4°C. Protein stock solution was filtered using 0.22 µm millipore syringe filter. The protein gave a single band during polyacrylamide gel electrophoresis. Concentration of the protein solution was determined experimentally using molar absorption coefficient, ε (M⁻¹ cm⁻¹) value of 9800 at 277.5 nm [21] (link). The concentrations of GdmCl and urea stock solutions were determined by refractive index measurements [22] (link). All solutions for optical measurements were prepared in the degassed 0.05 M cacodylic acid buffer containing 0.1 M KCl. Since pH of the protein solution may change upon addition of the osmolytes, pH of each solution was measured after the denaturation and refolding experiments. It was observed that the change in pH was not significant (∼0.02–0.04).

HPLC-grade water was obtained from a Mili-Q water purification system (Millipore Ltd., Bedford, MA, USA). HPLC-grade methanol (MeOH), HPLC-supergradient acetonitrile (ACN), sodium hydroxide (>99%) and reagent grade ammonium acetate (NH₄Ac) were obtained from Scharlab (Barcelona, Spain). Leucine-enkephalin (mass-axis calibration), formic acid (mobile phase modifier) and analytical-grade standards methionine sulfoxide and trimethylamine N-oxide were purchased from Sigma-Aldrich (Saint Louis, MO, USA).

Lysozyme (>0.95), L-proline (>0.99), 4-hydroxy-L-proline (>0.99), sarcosine (>0.98), trimethylamine N-oxide (>0.98) and thioflavin T (dye content: 0.65–0.75) were procured from Sigma-Aldrich Chemical Company USA. The listed purities of these compounds, on mass fraction basis, are given in the parenthesis. The solutions were prepared in deionized water which was double distilled and passed through Cole-Parmer mixed-bed ion exchange column. All the experiments were done in 40 mM phosphate buffer at pH 2.1 containing 100 mM NaCl. The stock solutions of Lysozyme were dialyzed overnight at 4°C against the buffer with at least three changes of the latter. The osmolyte solutions were also prepared in the final dialysate buffer. The concentration of the protein and ThT were determined on a Jasco V-550 uv-visible double beam spectrophotometer, using a value of A_{1 cm}^1% = 26.5 [19] at λ = 280 nm and E = 26,620 M¹ cm⁻¹ at λ = 412 nm [20] (link), respectively.