SEARCH

SEARCH BY CITATION

Keywords:

  • low field NMR;
  • β-lactoglobulin;
  • protein aggregation;
  • protein denaturation

Abstract

Low-field nuclear magnetic resonance (NMR) spin–spin relaxation (T2) measurements were used to study the denaturation and aggregation of β-lactoglobulin (β-LG) solutions of varying concentrations (1–80 g L−1) as they were heated at temperatures ranging from ambient up to 90 °C. For concentrations of 1–10 g L−1, the T2 of β-LG solutions did not change, even after heating to 90 °C. A decrease in T2 was only observed when solutions having higher concentrations (20–80 g L−1) were heated. Circular dichroism (CD) spectroscopy and fluorescence tests using the dye 1-anilino-8-naphthalene sulfonate (ANS) on 0.2 and 1 g L−1 solutions, respectively, indicated there were changes in the protein's secondary and tertiary conformations when the β-LG solutions reached 70 °C and above. In addition, dynamic light scattering (DLS) showed that protein aggregation occurred only at concentrations above 10 g L−1 and for heating at 70 °C and above. The hydrodynamic radius increased as T2 decreased. When excess 2-mercaptoethanol was added, the changes in both T2 and the hydrodynamic radius followed the same trend for all β-LG protein concentrations between 1 and 40 g L−1. These observations led to the conclusion that the changes in T2 were due to protein aggregation, not protein unfolding. Copyright © 2007 Society of Chemical Industry