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 improvement of a capillary electrophoresis apparatus. The improvement allowed both efficient control (temperature fluctuations <0.05 degrees C) and accurate measurement of the temperature (+/- 0.1 degrees C) within the capillary cartridge. CE allowed the thermodynamic parameters of beta-lactoglobulin thermal denaturation to be estimated. The transition temperature, Tm, 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 (deltaH, deltaS, deltaCp). Finally, the stability curve, i.e., the temperature dependence of the free energy change (deltaG) 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.
Rochu
D
D
Unité d'Enzymologie, Centre de Recherches du Service de Santé des Armées, La Tronche, France. rochudan@compuserve.com
Ducret
G
G
Ribes
F
F
Vanin
S
S
Masson
P
P
D000818:Animals; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000032:analysis*; D010710:Phosphates / Q000737:chemistry; D011489:Protein Denaturation; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D002021:Buffers; D007782:Lactoglobulins; D010710:Phosphates; C018279:sodium phosphate