α,ω-Bis-quaternary ammonium alkanes as effective buffer additives for enhanced capillary electrophoretic separation of glycoproteins

The egg white glycoprotein, ovalbumin, is known to be microheterogeneous as a result of its varied glycan content. The use of 1,4-diaminobutane (DAB) as a buffer additive has been shown to be key in the high-resolution capillary electrophoretic separation of "glycoforms" of this protein [Anal. Biochem. 205 (1992) 115]. Although a separation buffer consisting of 100 mM borate and 1 mM DAB allowed for adequate separation of ovalbumin glycoforms, prolonged separation times of 35-45 min were undesirable. In the present study, the α,ω-bis-quaternary ammonium alkanes, hexamethonium bromide (C₆MetBr), hexamethonium chloride (C₆MetCl) and decamethonium bromide (C₁₀MetBr) were tested as buffer additives for their effectiveness in the separation of ovalbumin glycoforms. Where 1 mM DAB gave optimal separation in ca. 45 min, 100 μM C₆MetCl or C₁₀MetBr yielded comparable resolution in less than 20 min. Results with the C₁₀MetBr were better than those obtained with C₆MetBr, indicating that there may be a correlation between effectiveness and alkyl chain length. Use of the chloride salt of C₆Met afforded the same resolution as the bromide salt in slightly shorter analysis time. The rank order for their effectiveness was found to be C₁₀MetBr > C₆MetCl > C₆MetBr > DAB. These results allow for speculation on the mode through which these additives exert their effect on resolution. Included in these are additive-wall coating interactions, protein-additive interactions, protein-wall interactions or any combination of these.