The molecular interactions between flavonoids and proteins play important roles in the biological fate and bioactivities of flavonoids. The objective of this study is to investigate molecular interactions between polymethoxyflavones (PMFs) and kappa casein. Two PMFs, nobiletin (NBT) and 5,3′,4′-tridemethylnobiletin (TDN), and two states of protein, native and thermal denatured, were used in this study. Surface enhanced Raman spectroscopy (SERS) and fluorescence spectroscopy were used to characterize and quantify the interactions, respectively. The SERS study shows that TDN has stronger binding to casein than NBT. SERS peak shifts assigned as OH bend in position C5 were observed when TDN interacting with casein. Additionaly, increased binding with thermal denatured casein(by heating at 95°C for 30 minutes) was observed for NBT. TDN, however, behaved in an opposite way, in which its binding affinity to thermal denatured casein decreased. Binding was also quantified by tryptophan quenching fluorescence spectroscopy, and quenching constant (Ksv) is calculated by the Stern–Volmer equation. The Ksv of NBT with casein before and after heating are 0.0478 and 0.0522 µM-1, while the Ksv of TDN with casein before and after heating are 0.1012 and 0.0592µM-1. Our results suggest that hydroxyl groups on PMFs played a key role in the interactions between PMFs and casein. NBT with no hydroxyl group interacts with kappa casein through hydrophobic interaction. Therefore, after casein denatured, increased interaction was observed due to the increased hydrophobic interaction. TDN with three hydroxyl groups interacts with kappa casein through hydrogen bonding and hydrophobic interaction, and hydrogen binding is the driving force. While heating destroyed the hydrogen binding sites of proteins with TDN, its interaction with denatured protein decreased. This study demonstrates the significance of the flavonoid structure and protein status on the flanoids-protein interactions.

Molecular characterization of the interactions between polymethoxylflavones and kappa casein

250th National Meeting (2015)

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