2.3. Iron and pH conditions
The assessment of stoichiometry was performed at four (patho)physiologically relevant pH values (4.5, 5.5, 6.8 and 7.5).
For the both lower pHs, 15 mM acetate buffers were used, while 15 mM HEPES buffer was used for pH 6.8. Because oxidation of Fe(II) significantly raises in the course of time at pH 7.5, hydroxylamine was added in the final concentration of 5 mM to the HEPES buffer in order to prevent Fe(II) oxidation. For the determination of Fe(III) chelation at pH 7.5, HEPES buffer without hydroxylamine was used. Two kinds of ferric solutions, i.e. ferric chloride hexahydrate
and ferric tartrate, were tested because of low solubility of Fe(III) at higher pHs. In this study, ferric chloride hexahydrate was used
at pHs 4.5 and 5.5 and ferric tartrate at pHs 6.8 and 7.5.