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Capillary zone electrophoresis with optimized temperature control for studying thermal denaturation of proteins at various pH
Author(s) -
Rochu Daniel,
Ducret Gilles,
Ribes Fabien,
Vanin Séverine,
Masson Patrick
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19990601)20:7<1586::aid-elps1586>3.0.co;2-3
Subject(s) - denaturation (fissile materials) , capillary electrophoresis , chemistry , electrophoresis , analytical chemistry (journal) , temperature control , chromatography , capillary action , dissociation (chemistry) , thermodynamics , nuclear chemistry , physics
Capillary electrophoresis (CE) was used to analyze the thermal denaturation of bovine β‐lactoglobulin at different pH. This model protein exhibits complex pH‐ and temperature association/dissociation dependence balances in its quaternary structure. The study was possible after modification and improvement of a capillary electrophoresis apparatus. The improvement allowed both efficient control (temperature fluctuations <0.05 o C) and accurate measurement of the temperature (± 0.1 o C) within the capillary cartridge. CE allowed the thermodynamic parameters of β‐lactoglobulin thermal denaturation to be estimated. The transition temperature, T m , was determined at acidic, neutral and alkaline pH. Van't Hoff analysis was performed through direct measurement of native and unfolded protein populations in the slow‐time regime. This allowed estimation of thermodynamic parameters (Δ H , Δ S , Δ C p ). Finally, the stability curve, i.e. , the temperature dependence of the free energy change (Δ G ) of protein unfolding was drawn. The accuracy of the parameters values compares with parameters obtained by calorimetric measurements. The available parameters and the requirement of minute amount of protein sample are of potential interest in the field of protein engineering and biological pharmaceuticals. Accordingly, CE can be proposed as a convenient tool to study protein stability and denaturation processes.