Thermally denatured proteins. Nuclear magnetic resonance, binding isotherm and chemical modification studies of thermally denatured bovine serum albumin

Abstract

The properties of thermally denatured bovine serum albumin have been investigated by n.m.r., binding isotherm and chemical modification studies. The marked reduction observed in the protein spin-spin relaxation times on thermal denaturation indicates that the protein chains become rigid. There was no change in the bulk water properties on thermal denaturation but a slight reduction in the amount of water bound to the protein was observed. The increase in the average water proton spin-spin relaxation rate on thermal denaturation results from a decrease in the bound water spin-spin relaxation time due to a reduction in the mobility of protein chains to which water molecules are bound. At temperatures higher than about 285 K, there is a contribution to the observed water proton relaxation times from exchange between labile protein protons and water protons, which results in a minimum in the spin-spin relaxation time at 328 K. The native structure of bovine serum albumin is not extensively disrupted on thermal denaturation. The results are consistent with a two step mechanism for thermal denaturation, involving aggregation of initially unfolded protein molecules. The initial unfolding involves mainly changes in the tertiary structure rather than in the secondary structure and aggregation results in the formation of a rigid network. The molecules are held together by co-operative forces involving many amino acid residues and, although intermolecular disulphide bonds do form on thermal denaturation of bovine serum albumin, chemical modification studies show that aggregation does not depend upon formation of chemical cross-links, as is often supposed.