Study of ethanol‐induced conformational changes of holo and apo α‐lactalbumin by spectroscopy and limited proteolysis

Abstract

This study was performed to contribute to the analysis of alpha-lactalbumin "molten globule" state by using spectral and proteolysis techniques. Samples of holo and apo alpha-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 alpha-lactalbumin indicated that its tertiary structure was maintained in 20% ethanol whereas it was altered in 30 and 40% ethanol. For apo alpha-lactalbumin, spectra were similar in all samples studied. Holo alpha-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 alpha-lactalbumin solutions. This study demonstrated that proteolytic enzymes are suitable tools to determine protein structure complementing physico-chemical studies.

Effect of high pressure on the structure and antibacterial activity of bovine lactoferrin treated in different media

“…conformation due to the absence of iron. A similar effect has been also observed for bovine α-la since the maximum of fluorescence intensity increases when calcium is removed from its native form (Wehbi et al 2006). The fluorescence of LFb did not show differences in λ max after HHP treatment with respect to the non-treated protein.…”

Effects of Hydrostatic High Pressure on the Structure and Antibacterial Activity of Recombinant Human Lactoferrin from Transgenic Rice

“…This effect has been observed also for -lactalbumin, because when the calcium bound to the native form of the protein was removed, the intensity of fluorescence increased. 38) These differences between the two forms of lactoferrin were also observed in a kinetic study of iron removal from bovine lactoferrin by EDTA, in which the increase in the intensity of fluorescence was used to measure the degree of iron removal. 39) The decrease in the intensity of maximum fluorescence reflects a conformational change in lactoferrin and corresponds to a decrease in solvent polarity that might be due to increased quenching of some tryptophan residues.…”

Obtaining antimicrobial peptides by controlled peptic hydrolysis of bovine hemoglobin

“…Therefore, several studies are show that protein hydrolysis in the presence of alcohol can permitted to induce a limited hydrolysis leading to peptidic population different from those obtain in the absence of alcohols [14][15][16]. Indeed, alcohols are commonly used to induce new structures or a partially unfolded state in proteins [17][18][19].…”