Protein stability function relations: native beta-lactoglobulin sulphhydryl-disulphide exchange with PDS

Intermolecular sulphhydryl-disulphide exchange with P-lactoglobulin dimer o ccurs when this dissociates to form monomers exposing two SH groups. This n otion is re-evaluated in the light of recent structural data suggesting tha t the degree of SH group exposure in beta-lactoglobulin is unaffected by di ssociation. beta-Lactoglobulin was treated with 2,2'-dipyridyl disulphide ( PDS). The rate of sulphhydryl-disulphide exchange was measured at sub-denat uration temperatures of 25-60 degrees C. Parallel studies were conducted by reacting PDS with reduced glutathione (GSH). The SH group of GSH was up to 31 000 times more reactive than beta-lactoglobulin. At pH 7 the reaction a ctivation enthalpy (Delta H-#) and entropy (Delta S-#) was 26 kJ mol(-1) an d -100 J mol(-1) K-1 respectively for GSH. For beta-lactoglobulin, Delta H- # was 157.2 kJ mol(-1) and Delta S-# was 254 J mol(-1) K-1. At pH 2.6, Delt a H-# was 14.4 kJ mol(-1) and Delta S-# was -213 J mol(-1) K-1 for GSH. The corresponding results for beta-lactoglobulin were 20.3 kJ mol(-1) and -147 J mol(-1) K-1. These and other thermodynamic results are discussed in term s of the effects of beta-lactoglobulin conformational structure and stabili ty on SH group reactivity. For native beta-lactoglobulin at neutral pH, int ermolecular sulphhydryl-disulphide exchange appears to involve the dissocia ted monomer. SH group activation probably arises from the lower structural stability of the monomer relative to the dimer. At pH 2.6 the mechanism of SH-disulphide exchange does not require protein dissociation and probably i nvolves breathing motions or localised changes in protein structure. (C) 20 00 Society of Chemical Industry.