High-performance concanavalin A affinity chromatography of liver and hepatoma membrane proteins.
Although the separation of water-soluble glycoproteins by high-performance (HP) concanavalin A (ConA) affinity chromatography (AC) is feasible, irregularities may be encountered with hydrophobic glycoproteins. The separation of plasma membrane glycoproteins from liver and Morris hepatoma 7777, used as a model, showed that not only the interaction between the lectin and the oligosaccharide portion of the glycoproteins plays a role in the chromatographic process, but also the hydrophobic interactions between sample and lectin and between sample and support. In this, the characteristics of the support, such as surface hydrophobicity and pore size, play an important part. It was found that a portion of the ConA is not covalently bound to the column, especially when elution is carried out with buffers containing detergents. Moreover, some extremely hydrophobic proteins could only be eluted from the column when high concentrations of detergents [1% (w/v) or higher] were applied. Despite these difficulties, four membrane glycoproteins from the liver with apparent molecular weights of 60, 80, 100 and 110-120 kilodaltons could be highly enriched by ConA HPAC. These proteins were further fractionated according to their strength of binding to the ConA and their different hydrophobic characteristics, using various detergents as eluents.
Josić
D
D
Institut für Molekularbiologie und Biochemie, Freien Universität Berlin, FRG.
Hofmann
W
W
Habermann
R
R
Reutter
W
W
D000818:Animals; D002021:Buffers; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000032:analysis; D008099:Liver / Q000032:analysis*; D008114:Liver Neoplasms, Experimental / Q000032:analysis*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification*; D008715:Methionine / Q000032:analysis; D009363:Neoplasm Proteins / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains
D002021:Buffers; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D003208:Concanavalin A; D005643:Fucose; D008715:Methionine