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In this study, we measured lactoferrin prepared in the presence of 70 (mol mol −1 ) ferric ions (70FeLf) by various physicochemical methods to create a structural model of 70FeLf. Structural stability of 70FeLf and that of native lactoferrin (Lf) was characterized by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy, revealing that 70FeLf was much more stable than native Lf. The shapes of 70FeLf and native Lf were observed directly by atomic force microscopy under various conditions. At pH 6.0, 70FeLf exists as a particle composed of about 15–16 Lf molecules. At pH 3.0, 70FeLf dissociates into native Lf, suggesting that non-covalent bonds between ferric ions and Lf are broken under acidic conditions. The structure of 70FeLf was also studied by ultracentrifugal sedimentation and 1 H nuclear magnetic resonance spectra. The state of ferric ions in 70FeLf was studied by electron spin resonance (ESR) spectroscopy. The ESR spectra of both 70FeLf and native Lf exhibited a signal at g = 4.0, while that of ferric ions in the free state exhibited a signal at g = 1.9, indicating that ferric ions in 70FeLf and native Lf exist in the high-spin state ( S = 5/2). Based on these results, we propose a structural model of 70FeLf, which is similar to that of casein micelle. Fe 3+ –HCO 3 − electrostatic interactions between Lf molecules stabilize the protein molecule, which may help the absorption of irons in vivo. |
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