Low‐field NMR: A tool for studying protein aggregation

Abstract

Abstract Low‐field nuclear magnetic resonance (NMR) spin–spin relaxation ( T 2 ) 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 T 2 of β‐LG solutions did not change, even after heating to 90 °C. A decrease in T 2 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 T 2 decreased. When excess 2‐mercaptoethanol was added, the changes in both T 2 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 T 2 were due to protein aggregation, not protein unfolding. Copyright © 2007 Society of Chemical Industry