Elucidating superoxide dismutase interactions with whey protein isolate using multi-spectroscopy, deep learning, and...

Superoxide dismutase (SOD) is an appealing additive for sports beverages owing to its antioxidant activity, while its thermal lability restricts its application in high-protein formulations. Here, we found that whey protein isolate increases the thermal stability of SOD by forming protein-protein complex. A Whey-protein isolate (WPI):SOD ratio of 5:1 resulted in the greatest enhancement of thermal stability, increasing half-life of SOD at 80 °C to 160.4 min, a 34.6% improvement over SOD alone. Multispectroscopic analyses revealed spontaneous, noncovalent, and single-site binding of SOD to WPI, which followed a Langmuir binding model (ΔG° = -22.19 ± 0.03 kJ/mol, K_d = 129.2 ± 1.19 μM). Deep-learning analysis showed SOD and β-lactoglobulin form a stable tetramer. Simulations confirmed this stability, driven by hydrophobic interactions and hydrogen bonds, is responsible for the complex's enhanced thermal resistance. Collectively, these results offer a practical strategy for improving SOD stability and provide a mechanistic basis for designing high-protein, antioxidant sports beverages.