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

The thiol group of β-lactoglobulin reacted very sluggishly with dithio-bis-nitro-benzoic acid as compared to that of glutathione at pH 6.85. The pK_app value 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° to 80°) 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, ΔG⁺₊, ΔH⁺₊, and ΔS⁺₊, for the heat-dissociation of β-lactoglobulin dimer were estimated to be 23 290 cal/mol, 31 160cal/mol, and 22.9 e.u. (at 70°), respectively. Addition of kosmotropic salts, chloride, tartrate, sulfate, phosphate, and citrate (0.2 m ) decreased the heat-induced exposure of the thiol group (at 70°), 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, Δ(ΔG⁺_+dimer), in the presence of the salts was positive, suggesting that these additives increase the stability of the dimer against heat. These salts also increased the conformational stability of β-lactoglobulin as revealed by an increase in - Δ(ΔG⁰_conf) values in their presence. Both Δ(ΔG⁺_+dimer) and - Δ(ΔG⁰_conf) 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.