In this study, we explore the effect of peroxidase-catalyzed cross-linking on the molecular conformation of apo-α-lactalbumin (apo-α-LA) and the resulting changes in protein surface hydrophobicity. In studying conformational changes, we distinguish between early stages of the reaction ("partial cross-linking"), in which only protein oligomers (10(6) Da > Mw ≥ 10(4) Da) are formed, and a later stage ("full cross-linking"), in which larger protein particles (Mw ≥ 10(6) Da) are formed. Partial cross-linking induces a moderate loss of α-helical content. Surprisingly, further cross-linking leads to a partial return of α-helices that are lost upon early cross-linking. At the same time, for partially and fully cross-linked apo-α-LA, almost all tertiary structure is lost. The protein surface hydrophobicity first increases for partial cross-linking, but then decreases again at full cross-linking. Our results highlight the subtle changes in protein conformation and surface hydrophobicity of apo-α-LA upon peroxidase-catalyzed cross-linking.
Keywords: enzymatic cross-linking; horseradish peroxidase; protein nanoparticles; structural transition; surface hydrophobicity; α-lactalbumin.