Lactoferrin supplied by the manufacturers was used without further purification. For apolactoferrin, preparation containing 50 mg/mL protein was dissolved in water and dialyzed extensively against 100 mM citrate buffer for 24 h, followed by dialysis against distilled water for 24 h [37 (link)]. Temperature (4 and 20 °C) and buffer pH (2.0–5.0) were modified in order to monitor their impact on iron desaturation. Iron saturation was calculated based on the A280/A466 ratio according to the calibration curve presented in “Results and discussion .” We define the iron saturation level of lactoferrin as the percentage of iron-binding sites occupied by ferric ions assuming that 2 mol of iron(III) ions is bound per 1 mol of protein. Thus, the given values refer to the percentage of differic lactoferrin. For some of these samples, the ICP-MS and ELISA tests were carried out and were used to prepare a calibration curve.
Hololactoferrin was prepared by the reaction of 50 mg/mL lactoferrin solution in 50 mM Tris–HCl, 150 mM NaCl (pH 7.4) with ferric nitrate salt in the presence of nitrilotriacetic acid (NTA) as well as different concentrations of sodium bicarbonate [24 (link)]. After incubation, excess iron was removed by dialysis against the same buffer solution without ferric salts for 24 h and against water for another 24 h. Various incubation times, temperatures, as well as ratios of Lf/Fe/NTA were employed to examine their effect on iron saturation efficiency; detailed conditions are depicted in the captions of figures.
Hololactoferrin was prepared by the reaction of 50 mg/mL lactoferrin solution in 50 mM Tris–HCl, 150 mM NaCl (pH 7.4) with ferric nitrate salt in the presence of nitrilotriacetic acid (NTA) as well as different concentrations of sodium bicarbonate [24 (link)]. After incubation, excess iron was removed by dialysis against the same buffer solution without ferric salts for 24 h and against water for another 24 h. Various incubation times, temperatures, as well as ratios of Lf/Fe/NTA were employed to examine their effect on iron saturation efficiency; detailed conditions are depicted in the captions of figures.
