Lectin Activity: Comparison
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Please note this is a comparison between Version 2 by Conner Chen and Version 1 by Clara Mariana Gonçalves Lima.

The purification of biomolecules with a high degree of specificity, such as lectins, has garnered interest in the use of fixed non-traditional beds functionalized with ligands of particular interest. The interaction is both robust enough to permit the adsorption of glycoproteins and reversible enough to permit the dissociation of molecules in response to changes in the solution’s pH. Studies on unconventional adsorbents, such as chromatographic supports, can substantiate, enrich, and assist projects in various areas of knowledge. Polyacrylamide cryogens are emerging and efficient, and can be synthesized and have their matrices modified for multiple purposes and chromatographic techniques. They are also functional and have low costs compared to conventional chromatographic adsorbents. In this context, lectins can mainly be used in the prevention of autoimmune diseases and in studies with biosensors.

  • purification of bio compounds
  • macromolecules
  • affinity chromatography

1. Introduction

Developing techniques and methods for separating and purifying biological macromolecules such as proteins has been an important prerequisite for many of the advances made in several areas of knowledge. In the field of chromatography, the development of new porous resin supports, new cross-linking agaroses, and new porous silicas allowed rapid growth in high-resolution techniques such as high-efficiency liquid chromatography, analytical and laboratory preparative scales, as well as for industrial chromatography in columns with several-hundred-liter bed volumes [1]. Macroporous monoliths, called cryogels, are produced from a solution of monomers and hydrophilic polymers using cryofreezing techniques previously used for biomedical applications. Cryogeleification is a specific type of synthesis of polymeric gels in which the formation begins in the cryogenic treatment of systems capable of forming gels [2]. The use of cryogels that incorporate free epoxy radicals on their surfaces is one of the most frequent ways to immobilize ligands of interest. This is due to the ease with which amine-, thiol-, or hydroxyl-group molecules can react to form a stable covalent bond. This is one of the most popular choices among the possibilities available. The glutaraldehyde (GLU) strategy has been highlighted as one of the other mechanisms likely to be employed with these reactive epoxy radicals. The essential advantage it offers is the ability to create a long spacer arm between the ligand and the adsorbing surface. Because of this, it can avoid the effect of steric hindrance, which can enhance the destabilizing power of the ligand as well as purify the target molecule. It is possible to purify lectins using macroporous adsorbents. Lectins are glycoproteins of immune origin which lack catalytic activity and have activity and specificity mediated by mono- and oligosaccharides; this method has a good chance of success. When different types of carbohydrates are immobilized, adsorbents with other properties are produced. Consequently, there is now a distinct possibility that these adsorbents can be used in processes intended to separate lectins [3,4][3][4].
The gel formation process in a semi-freezing zone allows the preparation of mechanically stable monolithic matrices composed of large interconnected macropores, since the pores formed in this process are 100 to 1000 times larger than those in other gels, which have pores in the range of 0.03–0.4 μm. This characteristic reveals the attractiveness and potential of these materials because the flow through these pores is purely convective and the resistance to mass transfer is low, which makes them permeable to aqueous solutions of proteins and even cell suspension [3,4,5][3][4][5].
Protein purification is grounded in studies of the physical–chemical characteristics, and structural and biological properties, being stimulated by its potential use in several areas of medicine, chemistry, biochemistry, and biology [6]. The most used technique to mediate such purification is affinity chromatography [7[7][8],8], based on highly specific and reversible interactions between pairs of biological materials (enzyme–substrate, enzyme–inhibitor, antigen–antibody) as well as studies using interactions with N-acetyl glucosamine [9], galactose derivatives [7], mannose [10], and proteins [11], among others, which can guarantee greater selectivity due to the specific stereochemical and typological characteristics presented by proteins. The principle of the method in question is to improve the separation capacity of biomolecules from specific, non-covalent bonds to insoluble supports, favoring the obtaining of bio-separations with high selectivity [12,13,14][12][13][14]. It is known that the use of monolithic, macroporous matrices applied in the purification of bio compounds is in constant growth and development [15].

2. Lectin Activity

Conceptually, lectins are proteins of non-immune origin that recognize and are associated with carbohydrates or glycoconjugates reversibly, with high affinity and specificity. Due to this ability, these biomolecules have important biological effects, such as insecticide, bactericidal, antitumor, and fungicide, in addition to an injunction on HIV-I protease, and became essential instruments in the diagnosis of diseases, identification of microorganism strains, and in studies related to blood types. Plant lectins have been used in cell biology and immunology as diagnostic and immunomodulatory agents, as well as for therapeutic purposes [16]. In addition, they can be used in the production of biosensors for the food industry, verifying the presence of microorganisms to ensure the quality of raw materials and industrialized products [17,18][17][18]. It is worth noting that the study of Matoba et al. [19] increased the antiviral activity of lectins. Such proteins have as characteristics the recognition and maintenance of specific and reversible bonds to mono- or oligosaccharides and other substances containing sugars, maintaining the covalent structure of these glycosidic ligands [19]. They can precipitate cells, glycoconjugates, and polysaccharides from animal, plant, virus, and bacterial sources [20,21][20][21]. The binding of lectins with sugars is attributed to a carbohydrate recognition domain (CRD) within their polypeptide structure. The interaction of lectins with certain carbohydrates can be as specific as the interaction between antigen and antibody or substrate and enzyme. Some are metalloproteins, in other words, they require the presence of metal cations at their specific binding sites with carbohydrates in connection with them, resembling metalloproteases; but lectins do not present catalytic activity [20,22][20][22]. Generally, lectins have at least two binding sites for carbohydrates, which allow cross-linking between cells through combinations with sugars on the surface or between sugars contained in macromolecules, justifying their ability to agglutinate particles and precipitate glycoconjugates. The lectin–carbohydrate interaction is due to covalent bonds, in which water molecules, associated with the polar group of proteins and also around the carbohydrate, are displaced (Figure 1). This modification results in the formation of new hydrogen bonding networks, which, together with van der Waals forces, stabilize this interaction [7,23][7][23].
Separations 10 00036 g001
Figure 1. Scheme illustrating the binding of lectin to the carbohydrate through the carbohydrate recognition domain. The carbohydrate–lectin interaction involves, among other non-covalent forces, the formation of hydrogen bonds and hydrophobic interactions.
The structure of proteins and their activity are strongly influenced by environmental factors such as pH and temperature, in addition to chemical factors such as the presence of ions [24]. Temperature is a considerable factor in the maintenance of the native activity of lectin hemagglutination. Singh (2013) working with lectin of Momordica charantia (MCJ) observed that at 30 °C their activity was maintained, however the increase in temperature to 55 °C reduced their hemagglutinating activity by 50%, and this percentage of activity was maintained for 12 min [25]. The total loss of lectin activity occurred at 65 °C, with no recovery, even after a temperature reduction, and this showed that the kinetics of inactivation of lectin hemagglutination activity at this temperature is an irreversible process. Similar to temperature, pH is a significant influencing factor in lectin activity. Hemagglutination tests with lectin MCJ showed that it is active at pH values between 3 and 11. The hemagglutinating activity of MCJ increases with an increase in pH, and the activity maximizes in the pH range of 5 to 8. With the increase in pH, the hemagglutinating activity of MCJ decreased, maintaining less than 50% of pH agglutination activity between 10 and 11 [25]. Most lectins require metal ions to present agglutinating activity, as is the case with lectins of legumes that require Mn2+ and Ca2+. Studies conducted by Sharon and Lis [26] with concanavalin A (ConA) indicated that, in an acid iced medium, there was a removal of metal ions from the ConA molecule, eliminating its ability to bind to carbohydrates. However, such a reaction can be circumvented with the addition of Mn2+ and Ca2+ ions in this order, considering that metals confer a high degree of structural stability to ConA, protecting lectins against heat inactivation [26,27][26][27]. On the other hand, these same ions at high concentrations are capable of agglutinating cells, as observed by Silva et al. [15] in their study on the influence of Ca2+ and Mn2+ ions for their hemagglutinating activity of cassava leaf lectin. Thus, to ensure that the agglutination is being mediated by lectin and not by excess ions, it is necessary to inhibit this activity with the addition of carbohydrates [15].

References

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