Differences in heat stability and ligand binding among β-lactoglobulin genetic variants A, B and C using 1H NMR and fluorescence quenching

• We found the heat stability of different ß-lactoglobulin isoforms to be A < B < C.
• Binding constants of retinol and epigallocatechingallate were tested.
• Lactoglobulin A had more binding sites than B and C for epigallocatechingallate.
• Upon heat denaturation all isoforms developed new binding sites.
• The backbone mobility of the isoforms influenced the ligand binding ability.

The structure of β-lactoglobulin (β-LG) is well characterized, but the exact location of binding sites for retinol and (−)-epigallocatechingallate (EGCG) is still a subject of controversy. Here we report that the genetic β-LG variants A, B and C have different numbers of binding sites for retinol (almost completely incorporated into the calyx), as well as for EGCG (exclusively bound on the surface), and β-LG A with the most binding sites for EGCG, which include Tyr20, Phe151 and His59. Upon heat related unfolding, new unspecific binding sites emerge, which are comparable in number and affinity for retinol and for EGCG, and in the three genetic variants A, B and C. The findings of our study provide new insights into the use of β-LG as nanotransporter.