An FTIR study of the complex melting behavior of alpha-lactalbumin

We have studied the thermal denaturation of hole alpha-lactalbumin (alpha-L A) using FTIR spectroscopy by monitoring the absorbance of protein bands in the secondary structure sensitive amide I region. In agreement with result s obtained by other techniques, a major cooperative melting transition is o bserved between 60 and 80 degrees C. This main transition is found to invol ve predominantly the unfolding of helical structures. Addition of excess Ca 2+ ions raises the T-m of the cooperative melting transition but does not c hange the protein substructures involved in the transition. In addition to the main transition, there are at least two other distinct melting processe s. One of them is gradual, is independent of excess Ca2+, and occurs throug hout the measured temperature range below the major cooperative transition. This melting process involves denaturation of alpha-LA's beta-hairpin. The other melting process is affected by the addition of excess Ca2+ and invol ves the denaturation of helical structures. In the absence of excess calciu m, this transition, occurring between 45 and 60 degrees C, strongly overlap s with the major cooperative melting. Our results indicate that the beta-ha irpin of alpha-LA is not directly stabilized by the bound calcium ion prese nt in the hole protein and melts independently from the rest of the protein .