ALPHA,OMEGA-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 berate 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 alpha,omega-bisquaternary ammonium alkanes, hexamethonium bromide (C(6)MetBr), hexamethonium chloride (C(6)MetC1) and decamethonium bromide (C(10)MetBr) 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 mu M C(6)MetCl or C(10)MetBr yielded comparable resolution in less than 20 min. Results with the C(10)MetBr were better than those obtained with C(6)MetBr, indicating that there may be a correlation between effectiveness and alkyl chain length. Use of the chloride salt of C(6)Met afforded the same resolution as the bromide salt in slightly shorter analysis time. The rank order for their effectiveness was found to be C(10)MetBr > C(6)MetCl > C(6)MetBr > 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.