Evaluation of capillary isoelectric focusing in glycerol-water media with a view to hydrophobic protein applications.
Capillary isoelectric focusing (CIEF) separations are usually performed with neutral coated fused-silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (eta = 945 mPa x s at 25 degrees C), known to help stabilize any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to using commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 v/v glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused-silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their pI with a good agreement between experimental and literature aqueous pIs. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase, a complex mixture of protein isoforms. The good concordance with the separation obtained by the conventional CIEF system indicated the adequacy of this new system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form.
Busnel
Jean-Marc
JM
Laboratory of Electrochemistry and Analytical Chemistry, ENSCP, Paris, France.
Varenne
Anne
A
Descroix
Stéphanie
S
Peltre
Gabriel
G
Gohon
Yann
Y
Gareil
Pierre
P
D005990:Glycerol / Q000737:chemistry*; D007525:Isoelectric Focusing / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D014783:Viscosity; D014867:Water / Q000737:chemistry*
D011506:Proteins; D014867:Water; D005990:Glycerol