Exploring the binding behavior mechanism of vitamin B₁₂ to -Casein and -Casein: multi-spectroscopy and molecular dynamic approaches

The aim of this study was to investigate the behavior interaction of alpha-Casein-B-12 and beta-Casein-B-12 complexes as binary systems through the methods of multiple spectroscopic, zeta potential, calorimetric, and molecular dynamics (MD) simulation. Fluorescence spectroscopy denoted the role ofB(12)as a quencher in both cases of alpha-Casein and beta-Casein fluorescence intensities, which also verifies the existence of interactions. The quenching constants of alpha-Casein-B-12 and beta-Casein-B-12 complexes at 298 K in the first set of binding sites were 2.89 x 10(4) and 4.41 x 10(4) M-1, while the constants of second set of binding sites were 8.56 x 10(4) and 1.58 x 10(5) M-1, respectively. The data of synchronized fluorescence spectroscopy at Delta lambda = 60 nm were indicative of the closer location of beta-Casein-B-12 complex to the Tyr residues. Additionally, the binding distance between B-12 and the Trp residues of alpha-Casein and beta-Casein were obtained in accordance to the Forster'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 alpha-Casein-B-12 and beta-Casein-B-12 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 alpha-Casein and beta-Casein in the presence of B-12 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 alpha-Casein and beta-Casein upon their binding to B-12 as the binary system. The experimental results that were obtained throughout the binding of alpha-Casein-B-12 and beta-Casein-B-12 complexes were confirmed by molecular modeling.