Noncovalent Polycationic Coatings for Capillaries in Capillary Electrophoresis of Proteins

The adsorption of proteins with net positive charges (pI > pH) on the walls of fused-silica capillaries is a common problem in the analysis of proteins by capillary electrophoresis. This paper explores the use of polycationic polymers as noncovalent coatings to limit this problem. The behavior of three sets of proteins was compared using uncoated and coated capillaries: (i) a protein charge ladder obtained by acetylation of lysozyme (EC 3.2.1.17); (ii) a protein charge ladder obtained by acetylation of carbonic anhydrase II (EC 4.2.1.1); (iii) a test panel of proteins with a range of values of molecular weight and pI. Four polycationic polymers were examined: polyethylenimine (PEI; MW_av = 15 000), Polybrene (MW_av= 25 000), poly(methoxyethoxyethyl)ethylenimine (MW_av = 64 000), and poly(diallyldimethylammonium chloride) (MW_av = 10 000). Detection of proteins with high pI was readily achieved using the first three of these polycationic polymer coatings but not with the poly(diallyldimethylammonium chloride). Examination of the stability of these coatings indicates that they are robust: the change in electroosmotic flow was less than 10% for 25 replications of the same separations, using capillaries coated with PEI or Polybrene. This study demonstrates that the charge ladder obtained by acetylation of lysozyme is a good model with which to test the efficiency of polycationic coatings. A study of the electrophoretic mobilities of the members of this charge ladder at pH 8.3 determined the effective charge of lysozyme (Zp(0) = +7.6 ± 0.1) and established the acidity of the α-ammonium group of lysozyme (pK_a = 7.8 ± 0.1). Results from the test panel of proteins suggest that protein adsorption is mainly driven by electrostatic interactions.