High-performance liquid chromatography with concanavalin A immobilized by metal interactions on the stationary phase.
Concanavalin A (Con A) was immobilized via metal interactions on macroporous, microparticulate silica support having covalently bound iminodiacetic acid functions (IDA-silica) chelated with Cu(II) at the surface. The amount of copper and of Con A in the column could readily be controlled by the conditions used for chelating the metal by IDA-silica and for immobilization of the lectin. The retention behavior of columns packed with the stationary phase did not change under a wide range of elution conditions, indicating no loss of immobilized lectin. However, the Con A proper could readily be removed from the column at pH 3.0 or together with Cu(II) by perfusion with EDTA at neutral pH. Columns containing Con A immobilized by this technique exhibited dual retention behavior for proteins, glycoproteins, and carbohydrates according to the pertinent glycan-lectin or protein-metal interactions. The glycoproteins, peroxidase and alpha 1-acid glycoprotein, were retained by the Con A moiety and eluted with eluents containing competing sugars, whereas the proteins, beta-lactoglobulin, alpha-chymotrypsinogen A, and ribonuclease A and B were retained by the chelated copper and were eluted and separated with eluents containing sodium chloride or borate. Binding constants of glycosides on the immobilized Con A were evaluated chromatographically and found to be one-third to two-thirds those reported in the literature on the basis of experiments in free solution.
el Rassi
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Z
Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520.
Truei
Y
Y
Maa
Y F
YF
Horváth
C
C
D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D003208:Concanavalin A; D003300:Copper; D006023:Glycoproteins / Q000032:analysis; D007098:Imino Acids; D007700:Kinetics; D011506:Proteins / Q000032:analysis; D012822:Silicon Dioxide
D002241:Carbohydrates; D006023:Glycoproteins; D007098:Imino Acids; D011506:Proteins; D003208:Concanavalin A; D012822:Silicon Dioxide; D003300:Copper; C008109:iminodiacetic acid