Ammonium ferrous sulfate hexahydrate

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.

Ammonium ferrous sulfate hexahydrate ((NH₄)₂Fe(SO₄)₂·6H₂O), equine spleen apoferritin in 0.15 M NaCl, ethanol (C₂H_5sOH), 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), and trimethylamine N-oxide (Me₃NO) were obtained from SIGMA-Aldrich (Saint-Louis, MO, USA); Coomassie brilliant blue from Fluka (Buchs, Switzerland); hydrochloric acid (HCl) from ITES (Vranov nad Toplou, Slovakia); potassium thiocyanate (KSCN) from Slavus (Bratislava, Slovakia); phosphoric acid (H₃PO₄) from Centralchem (Bratislava, Slovakia), and demineralized water.

All chemicals were of analytical reagent grade, and deionized water was used for the preparation of all solutions throughout the experiment. A stock standard solution of iron (II) at 1000 mg L⁻¹ was prepared by dissolving 0.7022 g of ammonium ferrous sulfate hexahydrate (Sigma-Aldrich, England) in water containing 1.0% (v/v) concentrated sulfuric acid (BDH, England); then, the volume was adjusted to 100 mL. The stock standard solution of iron (III) at 1000 mg L⁻¹ was prepared in the same way with 0.1 g of ferric chloride (BDH, England). A series of working standard solutions were prepared daily by diluting the stock standard solution to the desired concentrations. Acetate buffer (0.2 M, 500 mL) pH 4.8 was prepared by dissolving 7.21 g of sodium Acetate trihydrate (Carlo Erba, Italy) in water containing 2.72 mL of acetic acid (Carlo Erba, Italy).

Human serum albumin (HSA) fatty acid free and essentially globulin free (A3782), bovine serum albumin (BSA) fatty acid free and essentially globulin free (A7030), methyl vanillate, 2,4,6-tri(2-pyridyl)-S-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH), 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (pyranine), potassium persulfate, ammonium ferrous sulfate hexahydrate, warfarin, ibuprofen, dansylglycine, and phenylbutazone were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Stock solutions of warfarin, ibuprofen, phenylbutazone, methyl vanillate and its dimer dimethyl divanillate (10 mmol L⁻¹) were prepared in dimethyl sulfoxide. From the stock solutions, working solutions (1 mmol L⁻¹ or less) were prepared in 50 mmol L⁻¹ phosphate buffer at pH 7.0. HSA was dissolved in 50 mmol L⁻¹ phosphate buffer at pH 7.0 to give a 1 mmol L⁻¹ stock solution.