Lectins are ubiquitous in nature, and are found in all classes of organism. They are easy to detect and often to isolate. In addition, many are available from commercial suppliers. They are now obtainable also by recombinant techniques.
The classic, and still simplest, way to detect the presence of a lectin in a biological material is to prepare an extract from the material and examine its ability to agglutinate erythrocytes (Fig. 3.1) (Rüdiger, 1993). A more refined screening procedure is based on the ability of these proteins to precipitate polysaccharides (Goldstein, 1976) (Fig. 3.2) or glycoproteins. If a positive result is obtained, it is essential to show that the agglutination or precipitation is specifically inhibited by mono- or oligosaccharides, i.e., it is sugar specific (Fig. 3.1). Hemagglutination is commonly assayed by the serial dilution technique using erythrocytes from humans or rabbits. Occasionally erythrocytes that have been treated with trypsin or sialidase are employed, since such cells are often more sensitive to agglutination than the untreated cells (Fig. 3.3). Hemagglutination also serves to monitor and quantify the activity of lectins in the course of purification.
Because of the wide use of the agglutination reaction, it deserves some comments (Lis & Sharon, 1986). For agglutination to occur, the lectin must bind to the cells and form cross-bridges between them. There is however no simple relation between the amount of lectin bound and agglutination. Cases are even known where considerable amounts of lectin are bound to cells, without causing agglutination. This is because agglutination is affected by many factors, among them accessibility of receptor sites, membrane fluidity and metabolic state of the cells. It is also influenced by external conditions of the assay, such as temperature, cell concentration, mixing and so on. The relative contribution of the different factors depends on both the lectin and the cells examined. When agglutination does occur and it is inhibited by monoor oligosaccharides, it serves as an indication that carbohydrate structures for which the lectin is specific are present on the surface of the cell. Additional information on the nature of the receptors may be obtained with erythrocytes pretreated with enzymes, in particular glycosidases, or with sugar-modifying reagents, such as periodate. Agglutination with lectins is also of use in following changes on cell surfaces during physiological and pathological processes.
KeywordsHorseshoe Crab Hemagglutinating Activity Plant Lectin Recombinant Technique Serial Dilution Technique
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