Immobilization of sugars in supermacroporous cryogels for the purification of lectins by affinity chromatography.

Lectins are glycoproteins that bind to carbohydrates or glycoconjugates by specific interactions. The specificity of lectins to various carbohydrates is a determinant factor in the choice of ligand for the chromatographic matrix when using chromatography as a lectin purification technique. In this work, the immobilization of three different aminated carbohydrates on the surface of macroporous polymeric cryogels was evaluated. Carbohydrates were immobilized on cryogel surfaces via the glutaraldehyde method to create spacer arms, reducing steric hindrance. The immobilized N-acetyl-d-glucosamine and N-acetyl-d-mannosamine concentrations contained approximately 130mg of carbohydrate/g dehydrated cryogel, while the N-acetyl-d-galactosamine contained 105mg of carbohydrate/g dehydrated cryogel. Scanning electron microscopy showed that the physical structure and porosity of the chromatographic columns were not affected by the immobilization process, maintaining an elevated hydration capacity and the macroporous structure of the cryogels. Adsorption of concanavalin A on cryogels functionalized with N-acetyl-d-glucosamine (cryo-d-GlcNAc) was tested, as well as its reuse capability. After 5 cycles of use, cryo-d-GlcNAc was shown to be stable, with an adsorptive capacity of around 50mg/g. Carbohydrate immobilization in polyacrylamide cryogels was satisfactory, with promise for applications in lectin purification processes.

Development of supermacroporous monolithic adsorbents for purifying lectins by affinity with sugars.

Affinity techniques are frequently used to purify biocompounds, because of specific interactions observed in many cases. One example are the lectins, proteins connected in a reversible manner and specific to carbohydrates or sugar-containing molecules. Four different methods were investigated (epoxy, Schiff base, glutaraldehyde and ethylenediamine) to immobilize the carbohydrate N-acetyl-d-glucosamine (d-GlcNAc) on the surface of supermacroporous cryogels made for lectin purification. The glutaraldehyde method presented the highest immobilization capacity of d-GlcNAc (147.77mg/g), while the ethylenediamine method presented the lowest capacity (32.47mg/g). FTIR spectra analysis confirmed the presence of the immobilized carbohydrate. The cryogels containing d-GlcNAc immobilized by the different methods were characterized in terms of swelling capacity, degree of expansion, porosity and constituent fractions. Results showed that the activation methods did not affect the macroporous structure. Images obtained from scanning electron microscopy evidenced the presence of interconnected macropores in the structure of the cryogels produced. The cryogels presented even lower flow resistance in the permeability analysis. Finally, the cryogel modified by the glutaraldehyde method was used in the Concanavalin A lectin adsorption process, presenting an adsorptive capacity of 44.49mg/g and high stability after five cycles of use.