Thermal and Alkaline Denaturation of Bovine β-Casein

Abstract

The secondary structure of bovine β-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in β-casein in addition to a wide range of secondary structure elements, such as 10–20% α-helix, ∼30% turns, 32–35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78°C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40°C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (≤30°C) occur primarily in the monomer. It is hypothesized that the transition at 10°C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40°C are more likely the reflection of hydrophobic changes in the core of β-casein. As β-casein undergoes self-association and increases in size, the transition at higher temperature (78°C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that β-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.