Hydrophobic interactions in human casein micelle formation: β-casein aggregation

Summary

The association of non-phosphorylated (0-P) and fully phosphorylated (5-P) human β-caseins was studied by fluorescence spectroscopy and laser light scattering. The tryptophan fluorescence intensity (FI) level increased between 20 and 35 °C, indicating a change in the environment of that residue. A similar transition occurred when ANS was used as a probe. Transition temperatures were slightly lower in 10 mM-CaCl₂ but were not affected by an equivalent increase in ionic strength caused by NaCl. The magnitude of the FI change was less for the 5-P than the 0-P protein but was increased for both by CaCl₂ addition. These FI data were characteristic of a conformational change and this was supported by fluorescence polarization which indicated that with CaCl₂, tryptophan and ANS mobility increased at the transition temperature even though the extent of protein association also increased. Light scattering suggested that protein association proeeeded with the primary formation of submicellar aggregates containing 20–30 monomers which then associated further to form particles of minimum micelle size (12–15 submicelles), and eventually larger. The temperature of precipitation of the 5-P form in the presence of CaCl₂ was lower than the conformational transition and suggested that both hydrophobic interactions and Ca bridges between phosphate esters on adjacent molecules are important in micelle formation.