Micellization of Bovine β-Casein Studied by Isothermal Titration Microcalorimetry and Cryogenic Transmission Electron Microscopy

The association behavior, critical micellization concentration (CMC), and enthalpy of demicellization (ΔH demic) of bovine β-casein were studied, for the first time by isothermal titration calorimetry, in a pH 7.0 phosphate buffer with 0.1 ionic strength and in pure water. In the buffer solutions, the CMC decreased asymptotically from 0.15 to 0.006 mM as the temperature was raised from 16 to 45 °C. ΔH demic decreased with increasing temperature between 16 and 28 °C but increased from 28 to 45 °C. Thermodynamic analysis below 30 °C is consistent with the Kegeles shell model, which suggests a stepwise association process. At higher temperatures, this model exhibits limitations, and the micellization becomes much more cooperative. The CMC values in water, measured between 17 and 28 °C, decreased with increasing temperature and, expectedly, were higher than those found in the buffer solutions. β-Casein micelles were visualized and characterized, for the first time in their hydrated state, using advanced digital-imaging cryogenic transmission electron microscopy. The images revealed small, oblate micelles, about ∼13 nm in diameter. The micelles shape and dimensions remained nearly constant in the temperature range of 24−35 °C. Keywords: β-Casein; ITC; cryo-TEM; self-association; micellization