Concanavalin affinity chromatography

Purified by dissolving in Triton X-100 and deoxycholate, and by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose [Grondal and Zimmerman Biochem J 245 805 1987]. 

Affinity chromatography was carried out on columns prepared with lightly carboxymethylated chitin, which is known to be a poor substrate for lysozyme. Both native lysozyme and regenerated 13-105 were bound to the column at pH 7 and eluted at pH 3. As controls, the basic proteins cytochrome c and pancreatic RNase A, as well as concanavalin A and a-amylase, were not bound from the same solvent at pH 7. These findings constitute a third line of evidence for formation of native-like structure in regenerated 13-105. 

Instead of dialysis or gel filtration, an affinity chromatography on Concanavalin A Sepharose (cf. Protocol 3.6.2.4) is recommended, because on the one hand, no conjugated antibody is removed, and on the other hand, the sugar used for elution stabilizes the enzyme-antibody conjugate in solution. 

Nicollet, I., Lebreton, J. P., Fontaine, M., and Hiron, M. (1981) Evidence for alpha-1-acid glycoprotein populauons of different pi values after concanavalin A affinity chromatography. Study of their evoluuon during inflammauon in man. Biochtm. Biophys. Acta 668, 235-245. 

Laine, A, Hachulla, E., and Hayem, A (1989) The microheterogenelly of serum apantichymotrypsin revealed by interaction with concanavalin A in crossed immunoaffinoelectrophoresis and in affinity chromatography. Electrophoresis 10, 227-233. 

Figure 4.5. Affinity Chromatography. Affinity chromatography of concanavalin A (shown in yellow) on a solid support containing covalently attached glucose residues (G). 

Wade et al. [46] have compared the experimental band profiles of p-nitrophenyl-ff-D-mannopyranoside on silica-bonded Concanavalin A, obtained in affinity chromatography, and the best fit parameters to their model. This model i.e., Thomas model) uses a Langmuir kinetic and neglects the axial dispersion. The best values of the parameters are calculated using a Simplex program to minimize the sum of the residuals of the predided and experimental band profiles. Figure 14.10 illustrates the results obtained and shows excellent agreement. 

Figure 16.5 Affinity chromatography of peroxidase (after A. Borchert, P.O. Larsson and K. Mosbach, J. Chromatogr., 244, 49 (1982)]. Conditions sample, 4.1 ml of solution with 1 pg ml of protein column, 5cm x 5 mm i.d. stationary phase, concanavalin A on LiChrospher Si 1000, 10pm mobile phase, 0.05M sodium acetate, 0.5 M sodium chloride, 1 mM calcium chloride, 1 mM manganese chloride, pH 5.1 t shows pulse of 4.1 ml of 25 mM a-methyl-o-glycoside detector, solid line, UV, 280 nm broken line, visible, 405nm. Peaks = nonretained proteins this peak contains less than 2% peroxidase 2 = peroxidase.

Without doubt, concanavalin A (Con A) is the most celebrated and has proven to be one of the most useful of the plant lectins. Its physical chemical properties and carbohydratebinding properties are well documented in previous reviews [3,8]. Suffice it to note that it was first isolated and crystallized by Sumner and Howell in 1936, who showed it to require metal ions for its activity [74]. By virtue of its interaction with branched a-D-glucans, it is readily prepared by affinity chromatography on crossed-linked dextran (Sephadex) [75, 76]. A homotetramer at pH 7 (subunit M, = 26 500 Da) of Con A has been sequenced [77] and its crystal structure determined both in its native form [38,39] and complexed with methyl a-D-mannopyranoside [40] and Man(al-3)[Man(al-6)]Man[49]. 

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