Modifications Occur at Different Structural Levels During the Heat Denaturation of β‐Lactoglobulin

Abstract

Heat-induced modifications in the tertiary and quaternary structure of P-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 P-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 P-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, = 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 P-lactoglobulin at neutral pH is proposed which stresses the relevant role of a highly hydrophobic, molten-globule-like free monomer that has an exposed sulfhydryl group on its surface.Keywords: p-lactoglobulin ; heat denaturation ; sulfhydryl groups.The globular protein P-lactoglobulin is found in the whey fraction of the milk of many mammals. In spite of numerous physical and biochemical studies, its function is not clearly understood [ l , 21. The crystal structure of bovine P-lactoglobulin has been determined and shows similarities to the plasma retinol-binding protein and the odorant-binding protein [3, 41. This finding suggests that the role of P-lactoglobulin may be connected with transport or accumulation of lipid-soluble biological components [5, 61.Refolding of the tertiary structure of &lactoglobulin from the chaotrope-denatured form has been investigated extensively at low pH, where the association of monomers into multimeric forms is minimal [7, 81 and refolding conforms to the moltenglobule hypothesis of intermediate formation in protein folding/ unfolding 191. The high stability of P-lactoglobulin at low pH has been explained by the strong stabilizing action of the two disulfide bonds present in its tertiary structure [2, lo]. The free, highly reactive -SH group of Cys121 in each monomer has been shown to be involved in intramolecular and intermolecular disulfide interchange with other -SH groups in treated milk [I 1 -131.Despite the large amount of structura...

Unfolding Intermediate in the Peroxisomal Flavoprotein d-Amino Acid Oxidase

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