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 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 degrees to 80 degrees) 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 degrees), 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 degrees), 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 increase the stability of the dimer against heat. These salts also increased the conformational stability of beta-lactoglobulin as revealed by an increase in -delta(delta G0conf) values in their presence. Both delta(delta G++dimer) and -delta(delta G0conf) values followed the order, chloride less than tartrate less than sulfate less than phosphate less than citrate. These salts seem to manifest their structure-stabilizing effect by increasing both inter- and intramolecular hydrophobic interactions via changes in structure of water. Study holds ProTherm entries: 10375, 10376, 10377, 10378, 10379, 10380 Extra Details: 10 mM EDTA added in the experiment. beta-lactoglobulin; hydrophobic interactions; salt effect;,structural stability; thermodynamic stability; thiol reactivity
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:40 p.m.
|Number of data points||6|
|Proteins||Beta-lactoglobulin ; Beta-lactoglobulin|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O ionic:citrate: 0.2 M ; Experimental Assay: dG_H2O ionic:phosphate: 0.2 M ; Experimental Assay: dG_H2O ionic:sulfate: 0.2 M ; Experimental Assay: dG_H2O ionic:tartrate: 0.2 M ; Experimental Assay: dG_H2O ionic:chloride: 0.2 M ; Experimental Assay: dG_H2O ionic::|
|Libraries||Mutations for sequence LIVTQTMKGLDIQKVAGTWYSLAMAASDISLLDAQSAPLRVYVEELKPTPEGDLEILLQKWENGECAQKKIIAEKTKIPAVFKIDALNENKVLVLDTDYKKYLLFCMENSAEPEQSLACQCLVRTPEVDDEALEKFDKALKALPMHIRLSFNPTQLEEQCHI|