Heteroprotein complex co-aggregation of ovalbumin and ovotransferrin: structure formation and thermodynamics

Egg white proteins exhibit high thermal susceptibility to undesired aggregation, which limits their processing utility. In this study, the thermally regulated co-aggregation mechanism between ovotransferrin (OVT) and ovalbumin (OVA) was systematically investigated. Turbidity kinetics and particle size distribution results showed that the initial aggregation occurred at 55°C, induced by 4.3% disulfide cleavage and a 38.9% enrichment of β-sheet ( P < 0.05). Size exclusion chromatography results showed that this structural unfolding was mainly due to OVT molecules. The structurally activated OVT then specifically anchored the double cavities of OVA. Subsequently, upon heating at 60 - 65°C, the β-sheet reverted to the α-helix as detected by CD and FTIR spectroscopy, and the hydrophobic tyrosine and phenylalanine residues were reburied as detected by fluorescence spectroscopy, forming reversible metastable aggregates. At temperatures above 65°C, the larger heteroprotein complex (∼1.5 μm) emerged. ITC results showed that the co-aggregation was entropy-driven ( TΔS = +45.5 kJ/mol) with a stoichiometric ratio of 2:1 (OVT: OVA, n = 2.19 ± 0.12). Molecular docking and interaction force analyses revealed covalent disulfide bonds and multiple non-covalent bonds involved in this process. Notably, OVT acted as the aggregation initiator and governed the reverse aggregation, whereas OVA provided the structural backbone. These results provided a theoretical basis for the precise regulation of complex functional properties in the food industry.