Separation of alpha-acid glycoprotein glycoforms using affinity-based reversed micellar extraction and separation.
A new method for the preparation of the glycoforms of bovine alpha(1)-acid glycoprotein (AGP) is described relying on affinity-reversed micellar extraction and separation (ARMES). This method has proven effective in separating structurally similar glycoproteins and separating glycoproteins from nonglycosylated proteins from natural sources. In this method, individual glycoforms complex with the lectin, concanavalin A (ConA) are extracted into an organic-phase reversed micellar solution formed by Aerosol OT (AOT). The purity of three AGP glycoforms isolated was assessed by hydroxyapatite high-performance liquid chromatography (HPLC), gel-permeation chromatography and SDS-PAGE. The glycan structure of the pure glycoforms was analyzed. Oligosaccharide mapping using capillary electrophoresis (CE) and PAGE showed the glycans obtained from each glycoform to be distinctly different. ARMES can be used for the semi-preparative scale resolution of the glycoforms of bovine AGP or other therapeutic glycoproteins.
Choe
J
J
Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Zhang
F
F
Wolff
M W
MW
Murhammer
D W
DW
Linhardt
R J
RJ
Dordick
J S
JS
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002417:Cattle; D003208:Concanavalin A / Q000737:chemistry / Q000378:metabolism; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006801:Humans; D008823:Micelles; D009961:Orosomucoid / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D008823:Micelles; D009961:Orosomucoid; D011134:Polysaccharides; D020033:Protein Isoforms; D003208:Concanavalin A