Heat treatment of bovine alpha-lactalbumin results in partially folded, disulfide bond shuffled states with enhanced surface activity.
Prolonged heating of holo bovine alpha-lactalbumin (BLA) at 80 degrees C in pH 7 phosphate buffer in the absence of a thiol initiator improves the surface activity of the protein at the air:water interface, as determined by surface tension measurements. Samples after 30, 60, and 120 min of heating were analyzed on cooling to room temperature. Size-exclusion chromatography shows sample heterogeneity that increases with the length of heating. After 120 min of heating monomeric, dimeric, and oligomeric forms of BLA are present, with aggregates formed from disulfide bond linked hydrolyzed protein fragments. NMR characterization at pH 7 in the presence of Ca2+ of the monomer species isolated from the sample heated for 120 min showed that it consisted of a mixture of refolded native protein and partially folded protein and that the partially folded protein species had spectral characteristics similar to those of the pH 2 molten globule state of the protein. Circular dichroism spectroscopy showed that the non-native species had approximately 40% of the alpha-helical content of the native state, but lacked persistent tertiary interactions. Proteomic analysis using thermolysin digestion of three predominant non-native monomeric forms isolated by high-pressure liquid chromatography indicated the presence of disulfide shuffled isomers, containing the non-native 61-73 disulfide bond. These partially folded, disulfide shuffled species are largely responsible for the pronounced improvement in surface activity of the protein on heating.
Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, UK.
Ramani
R
Wijesinha-Bettoni
Chunli
C
Gao
John A
JA
Jenkins
Alan R
AR
Mackie
Peter J
PJ
Wilde
E N Clare
EN
Mills
Lorna J
LJ
Smith
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D007768:Lactalbumin / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D007768:Lactalbumin