Abstract
This study was performed to contribute to the analysis of α‐lactalbumin “molten globule” state by using spectral and proteolysis techniques. Samples of holo and apo α‐lactalbumin in the presence of different concentrations of ethanol were analyzed. Results of fluorescence spectroscopy of both forms showed that as ethanol concentration increased, the tryptophanyl residues became more accessible to the solvent. Near circular dichroism spectra of holo α‐lactalbumin indicated that its tertiary structure was maintained in 20% ethanol whereas it was altered in 30 and 40% ethanol. For apo α‐lactalbumin, spectra were similar in all samples studied. Holo α‐lactalbumin was resistant to trypsinolysis in 0% ethanol, whereas it was easily hydrolyzed in 20 and 30% ethanol. In the case of the apo form and in the absence of ethanol, 70% of the protein was degraded after 1 h. However, in the presence of 20 and 30% ethanol, the overall reaction rate was lowered. Peptides obtained after tryptic hydrolysis were identified by reversed‐phase high‐performance liquid chromatography coupled to mass spectrometry. Differences in population of produced peptides indicate the changes of folding intermediates present in the studied α‐lactalbumin solutions. This study demonstrated that proteolytic enzymes are suitable tools to determine protein structure complementing physico‐chemical studies.