Structural stability of β‐lactoglobulin in the presence of kosmotropic salts A kinetic and thermodynamic study

Abstract

The thiol group of .beta.-lactoglobulin reacted very sluggishly with dithio-bis-nitro-benzoic acid as compared to that of glutathione at pH 6.85. The pKapp vaue of the thiol group of the protein was 9.35. In the presence of 3 M urea, the thiol group reacted completely with dithio-bis-nitrobenzoic acid at pH 6.85. Heating (from 50.degree. to 80.degree.) increased the exposure of the thiol by dissociating the dimer unit. From the pseudo-first order rate constants of heat-exposure of thiol, thermodynamic activation parameters, .DELTA.G++, .DELTA.H++, and .DELTA.S++, for the heat-dissociation of .beta.-lactoglobulin dimer were estimated to be 23 290 cal/mol, 31 160 cal/mol, and 22.9 e.u. (at 70.degree.), respectively. Additon of kosmotropic salts, chloride, tartrate, sulfate, phosphate, and citrate (0.2 M) decreased the heat-induced exposure of the thiol group (at 70.degree.), probably by decreasing the dissociation of the dimer at pH 6.85. The relative change in free energy of activation for the dissociation of the dimer, .DELTA.(.DELTA.G++dimer), in the presence of the salts was positive, suggesting that these additives increse the stability of dimer against heat. These salts also increased the conformational stability of .beta.-lactoglobulin as revealed by an increased in -.DELTA.(.DELTA.GOconf) values in their presence. Both .DELTA.(.DELTA.G++dimer) and -.DELTA.(.DELTA.GOconf) values followed the order, chloride < tartrate < sulfate < phosphate < citrate. These salts seem to manifest their structure-stabilizing effect by increasing both inter- and intramolecular hydrophobic interactions via changes in structure of water.