**9.2 Metal ion requirements**

With a few exceptions, all lectins examined contain metal ions and in some cases evidence has been presented for the requirement of Mn2+ or Ca2+ (Emmerich et al., 1994) for activity (Table 2). Treatment with ethylene-diamine tetra acetic acid (EDTA) at neutral pH did not remove the metal ions from Concanavalin A (Doyle et al., 1984), soybean agglutinin (Jaffe et al., 1974) or lima bean lectin (Galbraith and Goldstein, 1970). Reversible removal of metal


Table 2. Metal content and metal requirements for activity of lectins

lectins. But lectins comprising of non-identical subunits are known as seen in soyabean agglutinin (Lotan et al., 1975) and the lectin from *Dolichos biflorus* (Carter and Etzler, 1975) which are tetramers, consisting of two types of subunits (Wright et al., 1996). A different type of subunit heterogeneity was first demonstrated in Concanavalin A (Abe et al., 1971; and Wang et al., 1971). The anti-B lectin from *Bandeiraea simplicifolia* consists of a family of five closely related proteins, each of which is a tetramer of one or two types of subunits. One of the subunits is specific for *N*-acetyl galactosamine, whereas the specificity of the other is confined to α-galactose (Goldstein and Hayes, 1978). The structure of *Bandeiraea simplicifolia*  isolectins is analogous to that of PHA isolectins. They have five tetrameric proteins comprising of varying proportions of two classes of subunits (Miller et al., 1973; Rasanen et al., 1973; and Leavitt et al., 1977). These subunits show difference in properties. It is assumed that it is due to their difference in the primary structure of subunits (Miller et al., 1973).

With a few exceptions, all lectins examined contain metal ions and in some cases evidence has been presented for the requirement of Mn2+ or Ca2+ (Emmerich et al., 1994) for activity (Table 2). Treatment with ethylene-diamine tetra acetic acid (EDTA) at neutral pH did not remove the metal ions from Concanavalin A (Doyle et al., 1984), soybean agglutinin (Jaffe et al., 1974) or lima bean lectin (Galbraith and Goldstein, 1970). Reversible removal of metal

> Lectin **Metal content (atom / mole) References Mn2+ Ca2+ Zn2+ Metal**

*Bandeiraea simplicifolia* I 1.2 2.0 Ca2+ Lescar et al., 2002 *Canavalia ensiformis* 4.0 4.0 Mn2+ Magnuson et al., 1983

*Datura stramonium* <0.2 <0.2 Kilpatrick, 1978 *Dolichos biflorus* 1.6 5.4 2.0 Etzler et al., 1970 *Euonymus europeus* 8.0 0.7 Petryniak et al., 1977 *Glycine max* 1.0-1.7 3.5-4.1 0.28 Mn2+ Lis and Sharon, 1973 *Lens culinaris* 0.64 3.8 Mn2+ Westbrook et al., 1984 *Marasrous oreades* 0.7 Winter et al., 2002 *Ononis hircina* 1.0 1.0 Horejsr et al., 1978 *Pisum sativum* 1.0 2.5 Ca2+ Reeke et al., 1986

*Phaseolus coccineus* 0.15 4.8 1.0 Perez-Campos et al., 1997

*Phaseolus lunatus* 1.0 4.0 Mn2+ Mach et al., 1991 *Phaseolus vulgaris* 0.24 6.2 Mn2+ Andrews, 1974 *Ricinus communis* <0.1 <0.1 <0.1 Mandal et al.,1989 *Sarothamnus scoparius* 1.5 0.8 Gurtler, 1978

*Ulex europeus* I 0.42 2.0 0.82 Sugii and Kabat, 1982 *Vicia cracca* 0.9 2.4 Sitohy et al., 2007

Table 2. Metal content and metal requirements for activity of lectins

**9.2 Metal ion requirements** 

ions can be achieved under acidic conditions. The Mn2+ in lectins can be replaced by a variety of transition-metal ions without loss of biological activity as demonstrated for Concanavalin A (Agrawal and Goldstein, 1968; and Shoham et al., 1973). Ca2+ in Concanavalin A could be replaced by Cd2+, but not by Ba2+ (Shoham et al., 1973). The metal ions confer a high degree of structural stability to Concanavalin A, protecting the lectin against heat inactivation and hydrolysis by proteolytic enzymes (Thomasson and Doyle, 1975). Ni2+ alone protects Concanavalin A against proteolysis at pH 7.0 but not at pH 8.2. Some lectins require metal ions for the saccharide-binding activity (Sumner and Howell, 1936). Extensive studies by NMR have revealed a complicated set of interlocking equilibrium involving the apoprotein and various complexes with metal ions and the saccharides (Brewer et al., 1983).

#### **10. Isolate and purification of lectin**

Purified lectins are essential for establish their molecular properties and are highly desirable for their many applications. In the past, lectins have been obtained solely from native sources, but they can now be produced also by recombinant techniques. Isolation of a lectin begins commonly with extraction of the tissue or organ in which it is present. This is simple in the case of plants, especially their seeds (Goldstein and Poretz, 1986; and Rudiger, 1993). The seeds are ground and the meal obtained is extracted with a neutral buffer. Often it is advisable to pre-extract the dry meal with an organic solvent, such as petroleum ether, to remove colored materials derived from the seed coat and lipids that may be present in large amounts. Animal tissues are either homogenized directly in the extraction buffer or the tissue is extracted first with acetone to remove water and lipids. The extraction buffer should preferably contain protease inhibitors to prevent degradation of the lectin during purification, and, in the case of membrane bound lectins, a detergent as well. Preliminary fractionation of the crude extract (e.g., by ammonium sulfate precipitation) is often done to obtain a protein fraction devoid of other constituents (e.g., polysaccharides in the case of plants). Final purification is achieved by affinity chromatography on a suitable adsorbent. A wide variety of affinity adsorbents, to suit any taste or purse, have been described in the literature and many of them can be purchased ready-made These include polysaccharides such as Sephadex, a polymer of glucose employed for the purification of Concanavalin A and pea lectin agarose (or Sepharose), a polymer of galactose, for the purification of the lectins from castor bean; acid-treated Sepharose for the purification of SBA; and chitin, a polymer of *N-*acetylglucosamine, for the purification of WGA. In the absence of readily available polysaccharides, use can be made of adsorbents consisting of carbohydrates or glycoproteins as such, or in the form of a synthetic derivative, that are covalently attached to an insoluble carrier. For instance, lactose coupled to Sepharose is the reagent of choice the purification of the lectins from peanut, eel electric organ or calf heartmuscle. *N-*acetylglucosamine bound to the same support serves for the purification of potato lectin and WGA, whereas immobilized porcine AH blood type substance is employed for the purification of the blood type A specific DBL and HPA. When working with lectins of an uncommon specificity, adsorbents have to be tailor made, as for example Sepharose bound asialoglycophorin for the purification of the blood type *N*-specific for lectin from *Vicia graminea*.

The lectin was purified from crude extract of mixer solution, commonly use chromatography technique such as, affinity chromatography, ion exchange

Antimicrobial Activity of Lectins from Plants 161

significant proportion of the amount ingested has reached the circulatory system with unimpaired hemagglutinating and immunological activities. In rodents, a diet containing lectins provoked intestinal and systemic immune responses to these proteins (Gomez et al., 1995). Furthermore, human serum was found to contain antibodies to the lectins of peanut,

Lectins are present abundantly in many plants. Despite this abundance, their precise biological roles in the plants to which they belong, are not well understood. The available

Lectins localized at the root hairs are the entry sites for rhizobia. The lectins then aggregate the rhizobia in the root nodules and make them immobile (Hamblin and Kent, 1973; Bohlool and Schmidt, 1974; Diaz et al., 1989; Brewin and Kardailsky, 1997; and Hirsch, 1995). Type specificity of host-parasite interactions between leguminous plants and particular strains of rhizobia infecting them is determined by lectins. The expression of the pea lectin gene in white clover roots enabled them to be nodulated by a rhizobium strain specific for the pea

Abrin, a type-II ribosome-inactivating protein (RIP), was the first lectin to be recognized as a defence protein (Peumans and Van Damme, 1995). Soon afterwards ricin also came to be recognized as a defence protein (Olsnes, 2004). Type-II RIPs which belong to the plant lectin family with β-trefoil fold are known to be toxic to animals and insects (Hartley and Lord, 2004; and Stirpe, 2004). Lectins from *Phaselous vulgaris* (PHA), *Robinia pseudocacia* and *Sambuscus nigra* have been reported to be toxic to higher animals (Peumans and Van Damme, 1995). Lectins from many plants, when ingested by animals, have resulted in toxic effects (Lis and Sharon, 1998), fungal growth in *Trichoderma viride* is inhibited by wheat germ aggiutinin (WGA) (Mirelman el al., 1975). Brambl and Gade (1985) have shown that eleven purified lectins, representing a wide spectrum of sugar specificity, inhibited the growth of fungal species *Neurospora crassa*, *Aspergilius amsteldomi* and *Botryodiplodia theobromae*. Known antifungal lectins include those which bind chitin (Peumans and Van Damme, 1995; Hirsch et al., 1995; Eijsden et al., 1995; Kijne, 1997; and Selitrennikoff, 2001). The anti-insect activity of many plants has been attributed to the presence of lectins in them. For example PHA (Chrispeels and Raikhel, 1991) pea nut agglutinin (PNA), WGA, *Maclura pomifera* agglutinin (MPA) and lectins from potato, thorn apple and osage orange show anti-insect activity against cowpea weevil. WGA and *Bauhinea purpurea* agglutinm are toxic to *Ostrinia nubilalis* larvae. Snow drop and garlic lectin show toxic effects on cowpea weevil and tobacco

The cell wall of bacteria not only precludes any interaction between the glycoconjugates on their membrane and carbohydrate-binding proteins but also prevents these proteins from

**12.1 Mediation of symbiotic relationship between nitrogen fixing microorganisms,** 

**12.2 Protection of plants from predatory animals and phytopathogens** 

hornworm (Hilder et al., 1995; and Peumans and Van Damme, 1995).

**13. Application to antimicrobial activity** 

soybean and wheat germ (Tchernychev and Wilchek 1996).

evidences suggest two main roles for them.

**primarily, rhizobia and leguminous plants** 

plant (Van Eijsden et al., 1995).

**12. Biological role** 

chromatography, and gel filtration chromatography. In 2004 had a research that used affinity chromatography to purify the lectin from human serum proteins by Concanavalin A sepharose column coupled to two-dimensional gel electrophoresis. The purified sample had 2 fractions before use this technique (Rodriguez-Pineiro et al., 2004). Next year, a lectin from the marine red alga *Gracilaria ornata* (*Gracilariaceae, Rodophyta*); GOL was purified by 2 steps chromatography technique consist of ion exchange chromatography on DEAE-cellulose and affinity chromatography on mucin-Sepharose 4B. The GOL significantly affected the development of *Callosobruchus maculatus* larvae, indicating the possibility of using this lectin in a biotechnological strategy for insect management of stored cowpea seeds. (Leite et al., 2005). In 2007 Shi et al. study lectin from raw and canned red kidney bean (*Phaseolus vulgaris*). They used gel filtration technique to purify. Use Affi-gel Blue gel sepharose compare to thyroglobulin-Sepharose to purify the lectin from red kidney bean. Found that the lectin from thyroglobulin more purify than Affi-gel Blue gel (Shi et al., 2007).

An alternative approach for the preparation of lectins has been made possible by the advent of recombinant DNA technology. It is based on the isolation of the cDNA or genomic DNA of the lectin, its insertion into a suitable vector and expression in an appropriate host cell. Isolation of the cDNA requires knowledge of at least part of the primary sequence of the lectin itself or of a structurally similar one. By this technique, several plant lectins, among them of pea (Stubbs et. al., 1986; and Van Eijsden et al., 1992), *Erythrina corallodendron* (Arango et al., 1993), peanut (Sharma and Surolia, 1994) and *Griffonia simplicifolia* (Zhu et al., 1996) have been expressed in *Escherichia coli*. Expression of plant lectins was also achieved in other systems, e.g. WGA in *Saccharomyces cerevisiae* (Nagahora et al., 1992), PHA and GNA in *Pichia pastoris* (Raemaekers et al., 1999), PNA in insect cells (Kumar et al., 1999) and SBA in monkey cells (Adar et al., 1997); (for a more complete listing of recombinant plant lectins) (Streicher and Sharon, 2003).
