Changes in the secondary structure of bovine casein by Fourier transform infrared spectroscopy: Effects of calcium and temperature

Bovine casein submicelles and reformed micelles, produced by addition of Ca 2+, were examined by Fourier transform infrared spectroscopy at 15 and 37 d egrees C in aqueous salt solutions of K+ and Na+. Previous measurements of caseins, made in D2O and in the solid form, can now be made in a more reali stic environment of H2O. When analyzed in detail, data obtained by Fourier transform infrared spectroscopy have the potential to show subtle changes i n secondary structural elements that are associated with changes in protein environment. Electrostatic binding of Ca2+ to casein resulted in a redistr ibution of the components of the infrared spectra. Addition of Ca2+ in salt solutions of K+ and Na+ led to apparent decreases in large loop or helical structures at 37 degrees C with concomitant increases in the percentage of structures having greater bond energy, such as turns and extended helical structures. At 15 degrees C, Na+ and K+ have differential effects on the Ca 2+-casein complexes. All of these observations are in accordance with the i mportant role of serine phosphate side chains as sites for Ca2+ binding in caseins and the swelling of the casein structure upon incorporation into re formed micelles at 37 degrees C. This new open, hydrated structure is buttr essed by a change in backbone as evidenced by a shift in absorbance to high er wave numbers (greater bond energies) as colloidal micelles are reformed.