Guidelines for the Preparative Fractionation of Human Serum Proteins on Gradient-Eluted Columns of Concanavalin A-Sepharose: Elution Positions of Fourteen Well-Characterized Proteins and Evidence for Concanavalin A-Reactive Albumin-IGA and -IGG Complexes

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Abstract
Systematic studies on the fractionation of serum proteins on gradient-eluted columns of concanavalin A[ConA)-Sepharose were carried out to determine if the oligosaccharide residues were sufficiently different to permit a reasonable separation and to determine where in the chromatogram these proteins would be eluted. Human whole serum and ammonium sulfate fractions derived therefrom were used in conjunction with 2.1 .times. 75 cm columns of ConA-Sepharose and a 4 .times. 400 ml gradient (Varigard) with 0.5 M methyl .alpha.-D-glucopyranoside as a limit buffer. The elution positions and chromatographic limits of 14 well-characterized human serum proteins were determined by double diffusion of aliquots of the effluent fractions (10X concentrated) in agarose gel against specific antibody and the general chromatographic distribution of the proteins by immunoelectrophoresis. Overall, the results demonstrate that the composition of the oligosaccharide side chain, like differences in molecular size, solubility and charge density, is a useful parameter in the chromatographic separation of proteins from serum. Although it is well-known that albumin is a nonglycoprotein, 1.0% of the protein showed that albumin binding was due to complex formation with IgA and IgG both of which possess the necessary complement of Con A-reactive residues for strong binding. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 2-mercaptoethanol-reduced albumin-IgA and -IgG complexes produced bands corresponding to the MW of albumin and the heavy and light chains of IgA and IgG whereas unreduced samples were not dissociated. When these complexes were reacted with ConA-Sepharose and treated with 2-mercaptoethanol, free albumin was eluted. The remaining adsorbed glycoprotein(s), IgA and IgG, could be eluted with methyl .alpha.-D-glucopyranoside. These results strongly suggest that these proteins and albumin are linked via a disulfide bond(s).