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

Abstract

The aim of this study was to investigate the behavior interaction of α-Casein-B₁₂ and β-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 β-Casein fluorescence intensities, which also verifies the existence of interactions. The quenching constants of α-Casein-B₁₂ and β-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 β-Casein-B₁₂ complex to the Tyr residues. Additionally, the binding distance between B₁₂ and the Trp residues of α-Casein and β-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 β-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 β-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 β-Casein upon their binding to B₁₂ as the binary system. The experimental results that were obtained throughout the binding of α-Casein-B₁₂ and β-Casein-B₁₂ complexes were confirmed by molecular modeling.

Communicated by Ramaswamy H. Sarma

Keywords:

• α-Casein
• β-Casein
• spectroscopy
• zeta potential
• circular dichorism
• MD simulation

Acknowledgements

The authors thank Dr. Ljungburg for the English editing.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

Data available on request from the authors. The data that support the findings of this study are available from the corresponding author, [JC], upon reasonable request.

Additional information

Funding

The financial support of the Research Council of the Mashhad Branch, Islamic Azad University is gratefully acknowledged.