Glycoproteomics meets thermodynamics: A calorimetric study of the effect of sialylation and synergistic anion on the binding of iron to human serum transferrin

Preparation and characterization of iron(iii) nitrilotriacetate complex in aqueous solutions for quantitative protein binding experiments

“…Sedimentation was observed with a 4 mM FeNTA solution in 0.1 M HEPES when 1 M NaOH was added to the buffer to adjust the pH aer the addition of the FeNTA stock solution. 33 In agreement with the authors of previous studies using NTA, we recommend preparing the FeNTA working solution immediately before use. However, by repeated UV-Vis measurements, we found that the 0.19 mM FeNTA solution (Fe : NTA = 1 : 2) is stable for several weeks even at room temperature.…”

“…Sedimentation was observed with a 4 mM FeNTA solution in 0.1 M HEPES when 1 M NaOH was added to the buffer to adjust the pH after the addition of the FeNTA stock solution. 33…”

“…The FeNTA solutions used for quantitative protein binding experiments ( e.g. the titration experiments with transferrin 33 ), were prepared by mixing the appropriate amount of the aqueous FeNTA stock solution with the appropriate buffer. The pH of the resulting solution was adjusted with 0.5 or 0.1 M NaOH and 1 or 5 M HCl.…”

“…We experienced this ourselves when working on the thermodynamics of the reaction of transferrin and FeNTA studied by isothermal titration calorimetry. 33 It is widely accepted that errors in syringe reactant concentration are a major source of variation in reported binding enthalpies. 34,35 Importantly, concentration errors, unlike procedural and baseline errors, do not lead to obviously pathological titration curves, but can still affect the tted thermodynamic results.…”