ABSTRACT
The aim of this study was to investigate the behavior interaction of α-Casein-B12 and ß-Casein-B12 complexes as binary systems through the methods of multiple spectroscopic, zeta potential, calorimetric, and molecular dynamics (MD) simulation. Fluorescence spectroscopy denoted the role ofB12as a quencher in both cases of α-Casein and ß-Casein fluorescence intensities, which also verifies the existence of interactions. The quenching constants of α-Casein-B12 and ß-Casein-B12 complexes at 298 K in the first set of binding sites were 2.89 × 104 and 4.41 × 104 M-1, while the constants of second set of binding sites were 8.56 × 104 and 1.58 × 105 M-1, respectively. The data of synchronized fluorescence spectroscopy at Δλ = 60 nm were indicative of the closer location of ß-Casein-B12 complex to the Tyr residues. Additionally, the binding distance between B12 and the Trp residues of α-Casein and ß-Casein were obtained in accordance to the Förster's theory of nonradioactive energy transfer to be 1.95 nm and 1.85 nm, respectively. Relatively, the RLS results demonstrated the production of larger particles in both systems, while the outcomes of zeta potential confirmed the formation of α-Casein-B12 and ß-Casein-B12 complexes and approved the existence of electrostatic interactions. We also evaluated the thermodynamic parameters by considering the fluorescence data at three varying temperatures. According to the nonlinear Stern-Volmer plots of α-Casein and ß-Casein in the presence of B12 in binary systems, the two sets of binding sites indicated the detection of two types of interaction behaviors. Time-resolved fluorescence results revealed that the fluorescence quenching of complexes are static mechanism. Furthermore, the outcomes of circular dichroism (CD) represented the occurrence of conformational changes in α-Casein and ß-Casein upon their binding to B12 as the binary system. The experimental results that were obtained throughout the binding of α-Casein-B12 and ß-Casein-B12 complexes were confirmed by molecular modeling.Communicated by Ramaswamy H. Sarma.
