Ferric ions release from iron-binding protein: Interaction between acrylamide and human serum transferrin and the underlying mechanisms of their binding

Acrylamide (ACR) is a surprisingly common chemical due to its widespread use in industry and various other applications. However, its toxicity is a matter of grave concern for public health. Even worse, ACR is frequently detected in numerous fried or baked carbohydrate-rich foods due to the Maillard browning reaction. Herein, this study intends to delineate the underlying molecular mechanisms of Fe ions released from iron-binding protein transferrin (TF) after acrylamide binding by combining multiple methods, including multiple complementary spectroscopic techniques (UV-Vis, fluorescence, and circular dichroism spectroscopy), isothermal titration calorimetry, ICP-MS measurements, and modeling simulations. Results indicated that free Fe was released from TF only under high-dose ACR exposure (>100 mu M). Acrylamide binding induced the loosening and unfolding of the backbone and polypeptide chain and destroyed the secondary structure of TF, thereby leading to protein misfolding and denaturation of TF and forming a larger size of TF agglomerates. Of which, H-binding and van der Waals force are the primary driving force during the binding interaction between ACR and TF. Further modeling simulations illustrated that ACR prefers to bind to the hinge region connecting the C-lobe and N-lobe, after that it attaches to the Fe binding sites of this protein, which is the cause of free Fe release from TF. Moreover, ACR interacted with the critical fluorophore residues (Tyr, Trp, and Phe) in the binding pocket, which might explain such a phenomenon of fluorescence sensitization. The two binding sites (Site 2 and Site 3) located around the Fe (III) ions with low-energy conformations are more suitable for ACR binding. Collectively, our study demonstrated that the loss of iron in TF caused by acrylamide-induced structural and conformational changes of transferrin.

Automated summary

This study elucidated the underlying molecular mechanisms of Fe ions released from TF after ACR binding, demonstrating that free Fe is only released under high-dose ACR exposure, inducing protein misfolding and denaturation of TF. The primary driving forces during the binding interaction between ACR and TF were identified as H-binding and van der Waals force, with ACR preferring to bind at specific sites on TF leading to Fe release.