Hydrogen–Deuterium exchange kinetics in β-lactoglobulin (−)-epicatechin complexes studied by FTIR spectroscopy

Abstract

Hydrogen–Deuterium exchange kinetics of β-lactoglobulin and β-lactoglobulin (−)-epicatechin solutions has been investigated through the analysis of the amide I absorption band at 1650cm−1 in the FTIR spectrum. H–D substitution in NH amides and residues of the protein results in a slight red-shift and in intensity changes of the amide I components: either these effects have been inspected in the framework of the Principal Components Analysis methods. The present analysis allowed to unveil three H–D kinetics at the timescale of the oligomeric fluctuations of the protein. A fast mechanism (lifetime from 5 to 10min) can be ascribed to the dynamics of protein oligomers and aggregates at the scale of the quaternary structure variations, and it is not observed in the complexes β-lactoglobulin (−)-epicatechin. The other slowest kinetics, whose lifetimes are in the range 1–10h, are here associated to dynamics of high-molecular weight complexes that hamper the proton exchange. The role of (−)-epicatechin as an enhancer of the formation of stable high-molecular weight aggregates from β-lactoglobulin is also discussed by comparison of the lifetimes at different protein concentrations.