Capillary zone electrophoresis with optimized temperature control for studying thermal denaturation of proteins at various pH

Capillary electrophoresis (CE) was used to analyze the thermal denaturation of bovine beta-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 im provement of a capillary electrophoresis apparatus. The improvement allowed both efficient control (temperature fluctuations <0.05 degrees C) and accu rate measurement of the temperature (+/- 0.1 degrees C) within the capillar y cartridge. CE allowed the thermodynamic parameters of P-lactoglobulin the rmal denaturation to be estimated. The transition temperature, T-m, was det ermined at acidic, neutral and alkaline pH. Van't Hoff analysis was perform ed through direct measurement of native and unfolded protein populations in the slow-time regime. This allowed estimation of thermodynamic parameters (Delta H, Delta S, Delta C-p). Finally, the stability curve, i.e., the temp erature dependence of the free energy change (Delta G) of protein unfolding was drawn. The accuracy of the parameters values compares with parameters obtained by calorimetric measurements. The available parameters and the req uirement of minute amount of protein sample are of potential interest in th e field of protein engineering and biological pharmaceuticals. Accordingly, CE can be proposed as a convenient tool to study protein stability and den aturation processes.