Control of α-Lactalbumin Aggregation by Modulation of Temperature and Concentration of Calcium and Cysteine

Research output: Contribution to journalJournal articleResearchpeer-review

Line R. Nielsen, Søren B. Nielsen, Zichen Zhao, Karsten Olsen, Jacob H. Nielsen, Marianne Nissen Lund

The effect of free cysteine (in different concentrations) on the thermal aggregation of calcium-saturated (Ca-sat) and -depleted (Ca-dep) alpha-lactalbumin (alpha-LA) was investigated at 25, 50, and 70 degrees C. The temperatures chosen were below the denaturation temperature (T-d) of Ca-dep and Ca-sat alpha-LA (25 degrees C), above the T-d of Ca-dep alpha-LA and below that of Ca-sat alpha-LA (50 degrees C), and above the T-d of Ca-sat alpha-LA (70 degrees C). Size-exclusion chromatography coupled to multiangle light scattering showed that no aggregation or only minor aggregation was obtained at the investigated temperatures for both Ca-dep and Casat alpha-LA even at extended holding times. Aggregates of Ca-sat alpha-LA were larger than those developed for Ca-dep alpha-LA. The addition of cysteine, a low-molecular-mass free thiol, resulted in increased aggregation of both Ca-sat and Ca-dep alpha-LA. Comparisons of SDS-PAGE run under reducing and nonreducing conditions showed that the formed cross-links were primarily disulfide bonds, but Western blots also showed small contributions from dityrosine cross-link formation. The aggregation kinetics related to monomer loss during heat treatment were determined by RP-UPLC and showed that the addition of cysteine increased the rate of aggregation. The activation energies for Ca-dep alpha-LA with 0.35 and 0.7 mM cysteine were found to be 59 +/- 1 and 46 +/- 4 kJ/mol, respectively, which showed that less energy was needed for the enhanced thermal aggregation of alpha-LA when the cysteine concentration was increased. This study showed that it was possible to control the aggregation size of alpha-LA by manipulating the incubation temperature and the cysteine concentration.
Original languageEnglish
JournalJournal of Agricultural and Food Chemistry
Volume66
Issue number27
Pages (from-to)7110-7120
Number of pages11
ISSN0021-8561
DOIs
Publication statusPublished - 2018

    Research areas

  • alpha-lactalbumin, calcium, cysteine, aggregation, thiol-disulfide exchange, kinetics

ID: 200819976