Study of conformational changes of ewe's holo (native) and apo-alpha-lactalbumin by spectroscopy and trypsinolysis

Conformational changes of ewe's alpha-lactalbumin (ALA) upon removal of Ca2+ were determined by surface hydrophobicity, calorimetry and circular dichroism. Native ewe's ALA resisted trypsinolysis, showing 4\% maximum degradation after 20 h of hydrolysis. Removal of bound calcium by addition of either ethylenediaminetetraacetic acid or ethylene glycol bis beta-aminoethyl ether-N,N,N,N-tetraacetic acid induced major protein conformational changes, enhancing its susceptibility to trypsinolysis, and leading to complete degradation of the protein. Reversed-phase high-performance liquid chromatography profiles of tryptic hydrolysate of Ca2+-free ALA were nearly the same through the whole enzymatic incubation period (24 h) showing the absence of sequential hydrolytic mechanism. They were characterized by the presence of five main peaks representing hydrophobic large-sized peptides. Cleaving the S-S bonds in the resulting hydrolysates with 2-mercaptoethanol gave rise to new peaks representing more hydrophilic and hydrophobic peptides.