High-performance capillary electrophoresis of glycoproteins: the use of modifiers of electroosmotic flow for analysis of microheterogeneity.
High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.
Landers
J P
JP
Department of Biochemistry and Molecular Biology, Mayo Clinic/Foundation, Rochester, Minnesota 55905.
Oda
R P
RP
Madden
B J
BJ
Spelsberg
T C
TC
D001881:Borates / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004586:Electrophoresis / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D009995:Osmosis; D010047:Ovalbumin / Q000737:chemistry; D010434:Pepsin A / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D011700:Putrescine / Q000737:chemistry
D001881:Borates; D006023:Glycoproteins; D010047:Ovalbumin; D010434:Pepsin A; D011700:Putrescine