Protein-carbohydrate interactions techniques

Once chemoenzymatically produced, these unnatural sialosides have demonstrated utility in a variety of biological applications. A sialoside consisting of 9-0-acetylated sialyl Tn antigen (sTn) was produced by one-pot synthesis and conjugated to human serum albumin to be used to evaluate the role of acetylation in carbohydrate-protein interactions. Para-nitrophenol-tagged sialosides can be used in a colorimetric assay to evaluate sialidase specificity. Biotin-tagged sialosides have been immobilized on a streptavidin-coated surface to study protein-carbohydrate interactions with surface plasmon resonance techniques. Size-defined... 

Many of these techniques have also been successfully utilized for the identification of inhibitors of protein-carbohydrate interactions. ELIS As are often employed, and their utility is illustrated in the efforts to identify selectin inhibitors [51-53]. However, other assays such as fluorescence polarization [33, 54] and carbohydrate affinity matrices [55] have also been utilized. These methods have provided valuable information about the substrate specificity of a number of carbohydrate-binding proteins and led to the discovery of many useful inhibitors. Still, there remains an acute need to develop effective high-throughput assays. 

Microarrays do not represent ideal formats for the examination of monovalent protein-carbohydrate interactions. Commonly, the densities of the immobilized oligosaccharides are too high to ensure that monovalent interactions are observed. Another limitation of the array technique is the requirement of purified receptor. Therefore, another more appropriate approach is needed to study interactions with cells. 

Techniques used to study protein-carbohydrate interactions... 

Discussion of the effect of ligand structure on protein-carbohydrate affinity requires an evaluation of complex stability constants. A munber of biophysical techniques are appropriate for the study of protein-carbohydrate interaction many of the more enlightening strategies are the topics of separate chapters elsewhere in this volume. We describe below three techniques used extensively in glycobiology— inhibition of hemagglutination, enzyme-linked lectin assay (ELLA), and isothermal titration microcalorimetry—and we consider the types of information provided by each technique in order to facilitate appropriate interpretation of the data. 

Similar click protocol was employed for synthesis of ferrocene-based triazolyl glycoconjugates 23 by reacting ferrocenyl azide 22 and terminal sugar-based acetylenic compound 21 under click condition. Resultant ferrocenyl glycoconjugate 23 is supposed to be applicable for the study of protein-carbohydrate interaction using electrochemical biosensing techniques (Scheme 6.7) [23]. 

Research work performed in the end of the last century by Haslam and co-workers showed that carbohydrates inhibit the protein-tannin interactions in solution (Luck et al. 1994 Ozawa et al. 1987). More recently, similar findings have been reported by de Freitas and co-workers using nephelometric techniques (Fig. 9D.12) for different carbohydrates and protein-tannin systems (Carvalho et al. 2006a, b de Freitas et al. 2003 Mateus et al 2004a). 

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