Modulation of Physicochemical and Conformational Properties of Kidney Bean Vicilin (Phaseolin) by Glycation with Glucose: Implications for Structure-Function Relationships of Legume Vicilins

The structure-function relationships of plant oligomeric globulins are still not fully recognized. The present work investigated the influence of glycation with glucose (at 1:50 and 1:100 protein/sugar molar ratios; incubation periods of 2.5, 5.0, and 10.0 h) on the physicochemical and conformational properties of kidney bean vicilin (phaseolin), with the aim of understanding the structure-function relationships of legume vicilins. Protein solubility (PS), surface charge (isoelectric point) and hydrophobicity (H-0), and secondary, tertiary, and/or quaternary conformations, as well as the emulsifying activities (emulsifying activity and emulsion stability indices, EAI and ESI) were evaluated. The 2.5 h incubation period of glycation led to least PS and highest H-0, and after that, the PS and H-0, on the contrary, gradually changed with increasing incubation period. The glycation increased the alpha-helix content and highly ordered secondary structures (alpha-helix + beta-strand), as evidenced by far-UV circular dichroism (CD) spectroscopy. Combined analyses of differential scanning calorimetry, intrinsic emission fluorescence, and near-UV CD spectroscopy indicated that phaseolin underwent a tertiary conformation unfolding and subsequent rearrangement process (to form a new tertiary conformation), whereas the quaternary conformational flexibility progressively increased upon increasing degree of glycation. The conformation rearrangement was more distinct at the 1:100 molar ratio than at the 1:50 counterpart. The glycation at 5.0 and 10.0 h periods considerably increased the EAI, but only at the 1:50 molar ratio was the ESI progressively increased with the incubation period. These results confirmed that besides surface properties (e.g., PS and H-0), the flexibility in tertiary and/or quaternary conformations played a major role in the emulsifying properties of glycated vicilins. The findings would have important implications for understanding the structure-function relationships of legume oligomeric globulins, thus providing a direction to further improve the surface-related functional properties of these proteins.