Frontal affinity chromatography of ovalbumin glycoasparagines on a concanavalin A-sepharose column. A quantitative study of the binding specificity of the lectin.
The interactions of Sepharose 4B-immobilized concanavalin A (ConA) with 10 glycoasparagines derived from ovalbumin were investigated quantitatively by frontal affinity chromatography. In this method, a carbohydrate solution is applied continuously to a ConA-Sepharose column and the retardation of the elution front is measured as a parameter of the strength of the interaction. The dissociation constant (Kd) for each saccharide with ConA can be determined. An analysis of the binding of p-nitrophenyl-alpha,D-mannoside has shown that the binding properties of ConA do not change essentially after immobilization on Sepharose 4B. Each of the ovalbumin glycoasparagines was labeled with tritium by the reductive methylation method for analysis. A comparison of the Kd values obtained showed that the binding of ConA varies considerably with very slight structural differences of the glycosyl chain. The results suggest that ConA recognizes a specific glycosyl chain structure, Man alpha 1-6(Man alpha 1-3)Man, in which at least one hydroxyl group at the C-3 position of C-6-linked mannose should be free. The glycoasparagines containing this structure bound strongly to ConA-Sepharose with dissociation constants below 3.4 X 10(-7) M.
D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D006020:Glycopeptides / Q000302:isolation & purification*; D008362:Mannosides / Q000032:analysis; D008433:Mathematics; D010047:Ovalbumin / Q000032:analysis*; D012685:Sepharose / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity
D006020:Glycopeptides; D008362:Mannosides; C028326:concanavalin A-sepharose; C007872:glycoasparagines; C002731:4-nitrophenyl-alpha-D-mannopyranoside; D010047:Ovalbumin; D012685:Sepharose