Covalent binding of food-derived blue pigment phycocyanobilin to bovine β-lactoglobulin under physiological conditions

•Phycocyanobilin (PCB) binds covalently to bovine β-lactoglobulin (BLG) via free Cys.•Binding is slow, of moderate affinity (Ka = 4 × 104 M−1), and stereo-selective.•The adduct has slightly altered secondary and tertiary protein structure.•Binding occurs under simulated gastrointestinal conditions.•Steered molecular dynamic puts PCB in the BLG calyx in the proximity of free Cys119. In this study, we investigated structural aspects of covalent binding of food derived blue pigment phycocyanobilin (PCB) to bovine β-lactoglobulin (BLG), major whey protein, by spectroscopic, electrophoretic, mass spectrometry and computational methods. At physiological pH (7.2), we found that covalent pigment binding via free cysteine residue is slow (ka = 0.065 min−1), of moderate affinity (Ka = 4 × 104 M−1), and stereo-selective. Binding also occurs at a broad pH range and under simulated gastrointestinal conditions. Adduct formation rises with pH, and in concentrated urea (ka = 0.101 min−1). The BLG-PCB adduct has slightly altered secondary and tertiary protein structure, and bound PCB has higher fluorescence and more stretched conformation than free chromophore. Combination of steered molecular dynamic for disulfide exchange, non-covalent and covalent docking, favours Cys119 residue in protein calyx as target for covalent BLG-PCB adduct formation. Our results suggest that this adduct can serve as delivery system of bioactive PCB.