Steric effects governing disulfide bond interchange during thermal aggregation in solutions of beta-lactoglobulin B and alpha-lactalbumin.
Intermolecular disulfide bond formation in pure beta-lactoglobulin (beta-Lg) B and in its 1:1 mixture with alpha-lactalbumin (alpha-La), heated at 85 degrees C for 10 min in solutions of low and high (100 mM NaCl) ionic strength and pH 6.0, was studied by reverse-phase HPLC and MALDI-TOF mass spectrometry. Disulfide bonding between beta-Lg monomers was more extensive than reported in the literature for a temperature of 68.5 degrees C, including formation of trimers connected by two of the three adjacent cysteines, C106/C119/C121. The participation of the different thiol groups in disulfide bonds appeared to depend on their location in the native structure, with surface-located cysteines more involved than internally located ones. This also applied to alpha-La-beta-Lg interactions, where the predominant participants were the surface-located alphaC111, alphaC120, alphaC61, and alphaC6. The least active participant was alphaC28, suggesting that it becomes sterically inaccessible during unfolding of the protein. High ionic strength apparently promoted disulfide bonding. The order of cysteine participation at the high ionic strength was similar to that at low ionic strength, with fewer native-location bonds observed and a lower activity of some groups, such as beta-C106/C119/C121 and alphaC61.
Livney
Yoav D
YD
Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Verespej
Edita
E
Dalgleish
Douglas G
DG
D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000737:chemistry*; D007782:Lactoglobulins / Q000737:chemistry*; D009994:Osmolar Concentration; D017510:Protein Folding; D012965:Sodium Chloride; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D007782:Lactoglobulins; D012996:Solutions; D012965:Sodium Chloride; D007768:Lactalbumin; D003545:Cysteine