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

Abstract

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 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., DTT) have been reported, this area is still understudied. Here, we provide new data on side reaction of the reducing agent TCEP in concentrated protein solution, suggesting that at high protein concentrations TCEP should be used with caution.

Controlled-Radical Polymerization of α-Lipoic Acid: A General Route to Degradable Vinyl Copolymers

“…To verify the presence of disulfide bonds along the backbone, poly­(butyl acrylate -co- α-lipoic acid) copolymers were degraded with tris­(2-carboxyethyl)­phosphine (TCEP, 1 equiv relative to disulfide content), a mild reducing agent commonly used in biochemical applications. , In a solution of THF/water (4:1) at 60 °C, the copolymer readily degrades into oligomers as evidenced by SEC (Figure and Table ). Alternative reagents are also capable of degrading disulfide bonds (e.g., NaBH 4 , Figure S14) and even the thioether bonds along the backbone are susceptible to degradation using AgNO 3 (Figure S15).…”

Interaction of Hyaluronan Acid with Some Proteins in Aqueous Solution as Studied by NMR

“…It can be supposed that such a change in the population of the gel component is associated with the manifestation of the lability of the gel formed by HA molecules. Such behavior, namely, limitation of diffusion of macromolecules in meshes formed by natural polymers, along with changes in the population of molecules involved in the formation of supramolecular structures, has already been observed [10,37].…”

Sodium caseinate as a particulate emulsifier for making indefinitely recycled pH-responsive emulsions

“…as 1 -casein is a triblock copolymer, and monomeric b-casein and k-casein have both hydrophobic and hydrophilic extremities. [23][24][25][26][27][28][29] NaCas, which is derived from casein and has a highly disordered structure, is a robust amphipathic protein that typically associates into spherical micelles (sub-micelles) with diameters in the range of 20-40 nm under neutral and weakly basic conditions (for example, pH 8.0) ( Fig. 1a 1 ).…”