Heat-induced aggregation of bovine lactoferrin at neutral pH: Effect of iron saturation

The goal of our study was to characterize the effect of iron saturation on the thermal aggregation of lactoferrin (Lf). Iron saturation markedly increased the thermal stability of Lf and decreased aggregation. Heating holo-Lf at 80 °C led to soluble polymer formation whereas apo and native Lf associated into large insoluble aggregates. The thermal aggregation of holo-Lf was mainly driven by non-covalent interactions, with intermolecular thiol/disulphide reactions also observed above 80 °C. For apo and native Lf, intermolecular thiol/disulphide reactions increased, suggesting that free thiol residues initiated cross-linking reactions and that iron saturation contributed to maintaining Lf disulphide bond integrity and protecting the protein from aggregation. We demonstrated that apo-Lf in the presence of N-ethylmaleimide (NEM) could undergo large aggregate formation through non-covalent interactions without the participation of intermolecular disulphide links. A mechanism for the thermal aggregation of Lf is proposed, emphasizing the influence of iron saturation.