Gonçalves Gabriel Ramos Ferreira, Gandolfi Olga Reinert Ramos, Santos Leandro Soares, Bonomo Renata Cristina Ferreira, Veloso Cristiane Martins, Veríssimo Lizzy Ayra Alcântara, Fontan Rafael da Costa Ilhéu
State University of Bahia Southwest, Itapetinga, BA, Brazil.
Federal University of Lavras, Lavras, MG, Brazil.
J Chromatogr B Analyt Technol Biomed Life Sci. 2017 Nov 15;1068-1069:71-77. doi: 10.1016/j.jchromb.2017.10.019. Epub 2017 Oct 9.
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.
凝集素是通过特定相互作用与碳水化合物或糖缀合物结合的糖蛋白。当使用色谱法作为凝集素纯化技术时，凝集素对各种碳水化合物的特异性是选择色谱基质配体的决定因素。在这项工作中，评估了三种不同的胺化碳水化合物在大孔聚合物冷冻凝胶表面的固定化情况。通过戊二醛法将碳水化合物固定在冷冻凝胶表面以形成间隔臂，减少空间位阻。固定化的N-乙酰基-D-葡萄糖胺和N-乙酰基-D-甘露糖胺浓度约为每克脱水冷冻凝胶含130毫克碳水化合物，而N-乙酰基-D-半乳糖胺每克脱水冷冻凝胶含105毫克碳水化合物。扫描电子显微镜显示，色谱柱的物理结构和孔隙率不受固定化过程的影响，保持了冷冻凝胶较高的水合能力和大孔结构。测试了伴刀豆球蛋白A在N-乙酰基-D-葡萄糖胺功能化的冷冻凝胶（冷冻-D-葡萄糖胺）上的吸附及其重复使用能力。经过5次使用循环后，冷冻-D-葡萄糖胺显示出稳定性，吸附容量约为50毫克/克。碳水化合物在聚丙烯酰胺冷冻凝胶中的固定化效果良好，有望应用于凝集素纯化过程。
