Effect of Reducing Agent TCEP on Translational Diffusion and Supramolecular Assembly in Aqueous Solutions of α‑Casein

The translational diffusion coefficient is highly sensitive to the size change of diffusing species and is ideally suited for the study of molecular association. Here, we used translational diffusion measurements by a pulsed-field gradient nuclear magnetic resonance (PFG NMR) technique to investigate the role of disulfide bonds in the formation of a supramolecular gel-like structure in the concentrated solution of α-casein. To reduce disulfide bonds, we added a commonly used reducing reagent tris­(2-carboxyethyl)­phosphine (TCEP) to α-casein solution. We found that the disruption of a disulfide bond Cys36–Cys40 in αs2-casein does not alter the translational diffusion or secondary structure of α-casein in dilute, 1 and 3% (wt %) solution. On the contrary, in concentrated, 15% (wt %) α-casein solution, in addition to the disruption of disulfide bonds, TCEP induced significant changes in gel properties. New long-lived intermolecular interactions formed, leading to the irreversible gel formation. While a few side reactions of TCEP (as well as other reducing agents, e.g., dithiothreitol) have been reported, this area is still understudied. Here, we provide new data on the side reaction of the reducing agent TCEP in concentrated protein solution, suggesting that at high protein concentrations TCEP should be used with caution.