Modifications occur at different structural levels during the heat denaturation of β-lactoglobulin

Heat-induced modifications in the tertiary and quaternary structure of β-lactoglobulin were followed at neutral pH for the protein at high temperature and for the protein that was heated and cooled. Fast changes in the environment of aromatic amino acids were apparent from near ultraviolet-CD spectra of the heated protein and their intensity increased with increasing temperature. These modifications were irreversible only at temperatures higher than 65-70°C. Addition of iodoacetamide during the heating/ cooling cycle greatly reduced the extent of irreversible modification of the tertiary structure of the protein. Reaction of the native β-lactoglobulin dimer with iodoacetamide or dithiobis (2-nitrobenzoic acid) was only observed upon heating at temperatures higher than 40°C and resulted in progressive reaction of the unique sulfhydryl group in each of the two protein monomers. The sulfhydryl reagents induced release of a monomeric protein species that was no longer able to aggregate to the native dimeric form or to sequentially form polymers as found in the protein after heating at high temperature. Dimer dissociation was identified as the rate-limiting step in the reaction of β-lactoglobulin with sulfhydryl reagents. It occurred at temperatures much lower than those required for appreciable modification of the tertiary structure of the protein, and had an extremely high activation energy (E(a) = 213 kJ/mol). These results are compared with other published data, and a general mechanism for the formation of early reactive Species in heat-treated β-lactoglobulin at neutral pi-I is proposed which stresses the relevant role of a highly hydrophobic, molten-globule-like-free monomer that has an exposed sulhydryl group on its surface.