Isolation and Characterization of Three Novel

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VI. Abstract. Lectins, the carbohydrate binding proteins, are biological molecules which interact ... their role as potential therapeutic agents that inhibit growth and agglutinate many ..... explained by the presence of special cell interactions that involve .... to identify ABO, H and N blood group systems of human (Sharon, 2007).

Isolation and Characterization of Three Novel Isolectins from the Medicinal Plant Tamarindus indica (Tamarind) Seeds

By:

Makarim El-Fadil Mohamed Osman

A Dissertation Submitted to Graduation Collage in Partial Fulfillment of the Requirements for the Master Degree in Science of Molecular Biology and Genetics.

Supervised By: Emad H.E. Konozy, PhD. Associate professor

Department of Zoology Faculty of Science Graduation Collage University of Khartoum 2008-2010

DEDICATIION

With great appreciation and respect, I dedicate this work To: Family... Friends... Class fellows... Makarim 2010

I

ACKNOWLEDGMENTS A special thanks to my supervisor Dr. Emad H.E. Konozy, Biotechnology park, Africa City of Technology, for making the dream a reality, making this project possible, keeping his promises to achieve a good piece of work and in recognition of his encouragement and assistance during every step throughout the project. Also regards are passed to Miss Amna Khalid for her continuous support and technical helps throw the project. We would like to acknowledge Dr. A. Alagib, and Ishtiag A. Abdelkareem, Tropical Medicine Research Institute, for providing materials and helping on running electrophoresis. We also thank Mr. Hassan. M. Saleem, Faculty of Agriculture, University of Khartoum, for supplying all bacterial strains. Finally we acknowledged all blood volunteers, and all laboratory assistants, Faculty of Science, University of Khartoum.

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Table of Contents Dedication....................................................................................................................................................... I. Acknowledgments..................................................................................................................................II Table of Contents....................................................................................................................................III Abstract..............................................................................................................................................................VI List of Tables.............................................................................................................................................VIII List of Figures.............................................................................................................................................IX

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEWS.............................................................................................................................1 1.1

Introduction.................................................................................................................1

1.2

Literature reviews...................................................................................................3

1.2.1 Lectins: The story of discovery................................................................................3 1.2.2 Lectin evolution and classifications........................................................................4 1.2.3 Natural functions of lectins.......................................................................................5 1.2.3.1

Intracellular lectins...................................................................................................5

1.2.3.2

Extracellular lectins..................................................................................................6

1.2.4 Lectins and biotechnology era.................................................................................8 1.2.5

Tamarindus indica the medicinal plant...............................................................10

III

CHAPTER TWO: MATERIALS AND METHODS....................12 2.1

Tamarindus indica seeds collection..............................................................12

2.2

Preparation of defatted acetone dried powder....................................12

2.3

Preparation of crude protein extract.........................................................12

2.4

Ammonium Sulfate fractionation and protein dialysis..................13

2.5

Protein and carbohydrate estimation........................................................14

2.6

Hemagglutination assay.....................................................................................14

2.7

Inhibition of hemagglutination assay........................................................15

2.8

EMtLs purification................................................................................................15

2.8.1 Affinity chromatography.........................................................................................15 2.8.2 Gel filtration.................................................................................................................15 2.8.3 Gel electrophoresis.....................................................................................................16 2.8.3.1

SDS-PAGE.........................................................................................................16

2.8.3.2

PAGE...................................................................................................................16

2.9

Lectin biophysiochemical characterizations.........................................17

2.9.1 Effect of pH on lectins activity...............................................................................17 2.9.2 Effect of denaturing agent on lectin activity.....................................................17 2.9.3 Effect of EDTA and metal ions on lectin activity............................................17 2.9.4 Effect of lectins on bacterial activity....................................................................18 2.9.4.1

Bacterial inhibitory assay................................................................................18

2.9.4.2

Bacterial agglutination assay..........................................................................18

CHAPTER THREE: RESULTS.....................................................................19 3.1

Protein extraction and fractionation.........................................................19 IV

3.2

Hemagglutination assay.....................................................................................20

3.3

Inhibition of hemagglutination assay........................................................26

3.4

Determination of molecular weights by gel filtration.....................27

3.5

Determination of protein subunits using SDS-PAGE.....................30

3.6

Effect of pH on lectins activity.......................................................................31

3.7

Effect of denaturing agent on lectins activity.......................................32

3.8

Effect of EDTA and metal ions on lectin activity..............................33

3.9

Bacterial inhibition and agglutination assay........................................34

CHAPTER FOUR: DISCUSSION AND CONCLUSIONS...35 4.1

Discussion...................................................................................................................35

4.2

Conclusion..................................................................................................................35

REFERENCES...............................................................................................................41 APPENDIX.........................................................................................................................47

V

Abstract Lectins, the carbohydrate binding proteins, are biological molecules which interact with glyco-conjugates in a reversible manner. Lectins attract great attention due to their role as potential therapeutic agents that inhibit growth and agglutinate many pathogenic and nonpathogenic agents, when interacting with their carbohydrate cell surface molecules differentially. Three variable lectins were precipitated from their solutions by ammonium sulfate saturation from seeds extract. Here forth denoted EMtL3, EMtL6, and EMtL8. These lectins exhibited different specificity towards human and rabbit erythrocytes. EMtL3, which obtained in the fraction precipitated with 30% (NH4)2SO4 saturation showed preference towards AB blood type, while EMtL6 was solely specific for A blood type. In contrast to MEtL3 and MEtL6, EMtL8 exhibited no human RBCs specificity however, could preferentially agglutinate rabbit RBCs. RBCs agglutination was appreciably enhanced upon treatment of RBCs by trypsin. On estimation of molecular weight by gel filtration EMtL3 had a molecular weight 130 kDa while EMtL6 had 11 kDa where as 130 kDa was reported for EMtL8 in pH 5 while in pH 7 the two proteins with molecular weight 130 kDa and 33 KDa were observed. Both EMtL3 and EMtL6 were glucose and N-acetyl glucosamine specific respectively. Interestingly EMtL8 was solely specific to maltose and mannose. The sugar specificity of both EMtL3 and EMtL6 allows using Sephadex G-150 as affinity chromatography. We noticed that the three isolectins did not inhibit the growth of Echerichia coli, Salmonella typhimurium, Shigella dysenteriae, Listeria monocytogenes and Staphylococcus aureus. However, they all agglutinated the human non-pathogenic E. coli with similar specificity. On the other hand, interestingly, EMtL6 agglutinated S. typhimurium and S. aureus. No agglutination occurred when lectins VI

incubated with Listeria monocytogenes and Staphylococcus aureus. Further studies on the effect of Temperature, pH, metal ions and denaturing agents on stability and activity of these isolectins had also been performed. In conclusion, the selective interaction of EMtL3, 6 and 8 with human ABO blood system as well as their preferential agglutinability of bacteria may throw some light on possible applications of these proteins in serology and drug delivery. Agglutination of E. coli, by all the three isolectins may open a frontier for a possible physiological involvement of these proteins in plant defense against relevant invader bacteria of the family Enterobacteriaceae. .

VII

List of Tables Table 1: Shows the amount of (NH4)2SO4 saturation needed to each fraction..........................................................................................................13 Table 2: Purification summary of the lectins from Tamarindus indica seeds..............................................................................................................24 Table 3: Estimation of protein and carbohydrates contents during different purification steps...........................................................................................25 Table 4: Minimum Inhibitory Concentration of carbohydrate needed to inhibit lectin activity.................................................................................................26

VIII

List of Figures Figure 1: Microscopic view of Hemagglutination of human O+ type....................20 Figure 2: Lectin specificity toward human erythrocyte suspension.......................21 Figure 3: Lectin activity determined by trypsin treated RBCs (250ug/ml in 100ml Tris-base pH8.0)............................................................................................22 Figure 4: The activity unit of the three lectins after incubation with trypsin treated and untreated rabbit erythrocyte suspension.................................................23 Figure 5: Gel filtration of crude extract, fractionated by (NH4)2SO4 (0-60%) saturation.......................................................................................................27 Figure 6: Gel filtration of fraction 80%, protein at pH7.0......................................28 Figure 7: Gel filtration of fraction 80% at pH5.0...................................................29 Figure 8: SDS-PAGE of purified lectins................................................................30 Figure 9: The effect of pH on lectin activity of EMtL3 and EMtL6......................31 Figure 10: Effect of the denaturing agent urea on EMtL3 andEMtL6 activity......32 Figure 11: Effect of metal ions (50mM) on EMtL3 and EMtL6 activity...............33 Figure 12: Microscopic view of a typical bacterial agglutination..........................34 Figure 13: Protein calibration curve (UV 280nm)......................................................49 Figure 14: Glucose calibration curve prepared using phenol-sulfuric acid method...........................................................................................................50 Figure 15: BSA calibration curve prepared using Lowry assay.............................50 Figure 16: Gel filtration molecular markers standard curve..................................51 Figure 17: Gel filtration profile of Frac30..............................................................51 Figure 18: Gel filtration profile of Frac60..............................................................52 Figure 19: Gel filtration profile of Frac80..............................................................52 Figure 20: Gel filtration profile of Frac80 at pH5.0................................................53

IX

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW 1.1. Introduction: Lectins, the proteins which are capable of agglutinating cells by their nonenzymatic ability of binding reversibly cell surface mono- or oligosaccharides without altering covalent structure of any glycosyl ligands (Moreira et al, 1993). This class of protein is ubiquitous in nature and being widely found in cells, membranes and secretomes of organisms of all kingdoms of life (Fik et al, 2000). This class of protein was known since the early of 19th century but, attracted little attention because it was assumed to be associated just with plant kingdom. However this idea started to be changed at the beginning of 1960s, when lectins used to study simple and complex carbohydrates architects and interacted with cell surface glycoconjugates, which helped to propel the field of glycobiology into the modern era (Sharon, 2008). Majority of lectins have high affinity binding toward certain type of sugars, they are able to distinguish between the different stereo-isomers of mono- and oligosaccharides. Unlike proteins, carbohydrates possess high degree of variation resulting from their monomers compositions with their carbon backbone length, side group orientation and substitution, moreover carbohydrate are often branched. So carbohydrates have that much complicated architecture, however, lectins can potentially recognize them with different affinity and specificity. This phenomenon enables lectins to agglutinate cells that are diverse in their plasma membrane structures or intracellular organelle carbohydrate coat (De Hoff, 2009). Although lectins are multi-domain proteins, the sugar binding activity often attributed to single protein module known as carbohydrate- recognition domain (CRD) (Dodd et al, 2001), the CRD possess high variation in amino acid sequence, which increases

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their specificity of binding sugars and rising them to compete immunoglobulin in their abroad variability to bind different antigens. Lectins bind carbohydrates by the multivalent system of binding, there is evidence that monovalent and bivalent binding event could occur at a single molecule level (Haworka et al, 2004). This enhances and increases the binding avidity of lectins to their ligands because of the presence of more than one CRD within the protein structure. Most of multivalent plant lectins arranged in dimmers or tetramers subunits, ranging usually in molecular mass from 25 to 35 kDa (Fik et al, 2000; De Hoff et al, 2009). Besides the natural functions that lectins play within the living system, they had been used widely in biotechnological field. Lectins have been used in serology to differentiate between the ABO system antigen determinants (Sharon, 2007). And because lectins are stable and survive digestion within the gastrointestinal tract, they could bind the epithelial cell and inter the blood circulation maintaining their full activity, they could be a good candidate for mediating drug targeting (Bies et al, 2003) or could be themselves a potent anticancer drugs (De Mejia et al,2005). In contrast, lectins could be used as a terrorist weapon, some of plant ricins have an anti-nutritional property, they could interfere with digestion and absorption and interrupt the macromolecules metabolisms also they could affect the bacterial flora and alter the immune state of the gastrointestinal tract (Audi et al, 2005, Vasconcelos et al, 2004).

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1.2 Literature review: 1.2.1 Lectins: The story of discovery: 1888, the year which was recorded in history of lectins discovery

by Peter

Hermann Stillmark, who extracted lectin from caster bean (Ricinus communis) and named it hemagglutinin or phytoagglutinin based on their plant origin, the materials which have ability to agglutinate cells, was highly toxic and considered antigenic by Ehrlich, the father of immunology, who named them ricin and use them as an antigenic model in his immunological studies, which enabled him to establish many fundamental concepts of immunology (Sharon and et al, 2004). However these plant materials were generally considered non-specific materials able of agglutinating animal red blood cells irrespective to blood type and animal species, but during the years 1948 and 1949, Renkonen and Boyed undergoes a systematic screening of hundreds of plant seed’s hemagglutinin to discover that each protein had specific activity toward certain type of erythrocyte, Boyed then in 1963, named the hemagglutinin materials, lectins (Osawa, 1989). At the beginning of 1960s, lectins became not anymore a protein found exclusively in plants (Sharon, 2008), the idea extended to cover the animal kingdom. When the phenomenon of reaggregation of dissociated marine sponge cells observed, and explained by the presence of special cell interactions that involve carbohydrates. By time more evidence for presence of lectin among invertebrates had been emerged and few of animal lectins from eel, snails and horseshoe crab had been isolated (Varki, 1999). In 1970s, the introduction of affinity chromatography technique for lectin purification increased the number of isolates dramatically. Lectin discovery expanded to include mammalian cells, when their function as a recognition molecules confirmed by the isolation of galactose- specific liver lectin during 1974 under the investigation of the mechanisms that control the life time of

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glycoprotein in blood circulation which is uptake by galactose- specific liver lectin and degraded in liver (Sharon, 2007). Lectins attracted more attentions with the demonstration that lectins are useful tools for investigating cell surface carbohydrate and when used for detecting, isolating and characterizing glycoconjugates and glycoproteins, declaring by that the new era of glycobiology (Sharon et al, 2004).

1.2.2 Lectin evolution and classification: Lectins have different classification among plant, animal and microbes. Plant lectins are concentrated in seeds more than any other tissues, although they were reported in leaves, roots and fruits, but with lower concentration. Basal expression levels of plant lectins are available now with the emergence of micrroarray technique that provides high throughput data of mRNA expression profile, these data shows that level of lectin expression varies among organ structure and changes under environmental and microorganismal stimuli (De Hoff et al, 2009). Structurally mature plant lectins classified into merolectin (Hevein) which contains single carbohydrate recognition domain (CRD), hololectin (ConA) the one that build up from at least two CRDs which may be identical or very homologous, that bind identical or structurally similar sugar, the other group is chimerolectins (Ricins) that characterized by the association of one or more CRDs arrayed randomly to unrelated domains, and finally the superlectins (TXCL-1) that consist restrictedly of at least two CRDs. (Liu et al, 2010). Previously, plant lectins were grouped in four families according to their CRDs into legume lectins, chitinbinding lectins, type-2 ribosome inactivating proteins and the monocot mannose binding lectins. Although their classification is still evolving and not all plant lectins can be classified into those four groups, more groups were emerged to locate most of them under the umbrella of major seven families which are legume 4

lectins, jacalin- related lectins, amaranthins, cucurbitacea phloem lectins, mannosebinding lectins and type-2 ribosome inactivating lectins (De Hoff et al, 2009). At the same time animal lectins undergo different way of classification based on their amino acid sequences and evolutionary relatedness, basically animal lectins perform eight well established groups which are categorized into two main branches, intracellular lectins: calnexin family, M- type, L- type and P- type which are located in luminal compartments of the secretary pathway, and the extracellular lectins: C- type, R-type, siglecs and galectins, the families which are either secreted into the extracellular or body fluid or localized to the plasma membrane. All comprehensive studies on animal lectin evolution have pointed to the intracellular lectins as the ancestral of animal lectins which appeared at an early stage of the eukaryotic cell evolution, suggesting that their functions are basic for cells in all organisms (Dodd, 2001). In contrast, microbial lectins classified according to their extracellular function to adhesins that were described by Nathan Sharon in 1970, hemagglutinin which first discovered in influenza virus in 1950 and toxins the protein produced by bacteria and action depend on glycan- binding subunit that allow toxin to join membrane glycoconjugates and deliver the functionally active toxin across the cell plasma membrane (Esko et al, 2009).

1.2.3 Natural function of lectins: 1.2.3.1

Intracellular lectins:

Although lectins have different classification among different organisms lineage, their functions are depend mainly on their cellular localization. Intracellular lectins generally function as chaperons dealing in glycoprotein sorting, trafficking, degradation and enzyme targeting. Lectins which display common physiochemical 5

characteristics which are shared between different species of the same genera suggest conserved roles in maintaining the internal environment of the organism (Konozy, 2002). For example, calnxein and calreticulin are lectins responsible for quality control of endoplasmic reticulum glycoprotein; they bind terminal glucose residues on the N- linked oligosaccharides and retain the misfolded glycoproteins back in the ER, the machinery by which they work are present in the early unicellular eukaryotes, the common progenitors of plants, animals and yeast. The other example is L-type lectin of plants and animals, named L-type after legumes, where they are abundant in seeds. This type of protein involves in protein sorting in luminal compartments of animal and plant cells. Mannose-6-phosphate lectin of plants also designated P-lectins in animals are trafficking lectins that bind mannose-6-phosphate of the glycoconjugates and direct them from Golgi apparatus into the lysosomes, their final destination to be degraded. One of the interesting proteins are animal mannose receptors that contains ricin CRD or R-type domain that binds N-acetylgalactosamine of circulating hormones mediating their clearance (Dodd et al, 2001). Lectins have shown to possess a modulating effect on several enzymes, for instance endogenous α-mannosidase activity was shown to increase in the presence of endogenous lectin by 35% (Kestwal et al, 2007). . 1.2.3.2

Extracellular lectins:

Extracellular lectins mediate all non-structural functions of the cell interactions starting from cell to cell or cell to matrix recognition events ending with defense events (Dodd et al, 2001) Mainly plant lectins which are targeted extracellularly or secreted assumed to function in deposition of storage proteins, maintaining seed dormancy, transporting carbohydrate, stimulation of embryonic plant cells mitogenically, elongation of cell wall, recognition of pollen, defense against pathogens and animal predators and in 6

symbiosis (Sharon et al, 2004). The hypothesis that plant lectins act in protection against pathogens comes from many experiments on wheat germ agglutinin (WGA), soybean lectin (SBL) and other lectins which inhibits sporulation of many fungal species. Another assumption was for the role of lectins in nitrogen fixation by legumes, where lectins bind carbohydrate of the lipopolysaccharides of the bacterial surface that fix nitrogen such as Bradyrhizobium sp. but not other symbionts. This provides two functions: bacterial adhesion and then nitrogen fixation (Sharon et al, 1987). Extracellular animal lectins synthesized either as free or membrane bound molecules function mainly during defense events. Lectins function mainly in discrimination between self and non-self cells during the phase of innate immunity of both vertebrates and invertebrates (Fujita, 2002). Free lectins that secreted in circulatory system survey the presence of the pathogens, the most common one is mannose binding lectin MBL that binds range of sugars including Nacetylglucosamine, mannose, N-acetlymannosamine, fucose and glucose (Turner, 2003) that present in the cell surface of the pathogen, this triggers series of proteolyic events associated with complement system defense of the innate immunity that ends with pathogen degradation (Male et al, 2006). At the same time bounded lectins like selectin mediates the homing of lymphocyte to the secondary lymphoid organs and to the site of inflammation. Intelectin plays another function beside its role in the innate immunity; this protein provokes the internalization of the sperm into the oocyte during the embryogenesis (Varki et al, 2009). Hemagglutination, adhesion and toxicity are process occur duo to the expression of lectins that causes pathogencity during the course of infection by mirobes. Hemagglutinin the viral lectin recognize specific carbohydrate architecture displayed at cell surface of the specific tissue, thus, the specificity of hemagglutinin determine the tropism of the pathogen in respect to the species and 7

tissue specificity. Adhesin, the filamentous lectins that called fimbrae or pili are located at the cell surface of the bacteria and mediates the pathogen- host interaction by the binding host glycolipids and glycoprotein (Sharon, 1987). The relationships between microbe and its host not always pathogenic, but also symbiosis often observed, when symbionts induces its host to express certain type of carbohydrate either to attach to it by its adhesins or to use it a source of carbon. Beside the extracellular bounded lectins, free lectins are produced and secreted by the microbes, called toxins that binds host cell membrane glycoconjugates and transfers its toxic subunit across the membrane (Varki et al, 2009).

1.2.4 lectins and biotechnology era: The discovery of lectins had transferred the field of glycobiology into new era, many lectins have been incorporated into affinity columns allowing the study and purifications of many glycoproteins, the most famous lectin affinity columns are Concanavalin A (Con A) which binds α-D-mannose and α-D-glucose, more over affinity lectins are used in clinical field to purify and separate many enzymes (Hage, 1999). The ability of lectin to distinguish between different carbohydrate structures that present on cell surface enables researchers to use them in serology to identify ABO, H and N blood group systems of human (Sharon, 2007). At the end of the last century, lectins had been used in drug delivery to overcome the poor biopharmaceutical drug properties such as limited water solubility and membrane permeability, lectins enhance those properties by increasing

the

residence time at the biological barriers, enhance drug concentration and permeability. Many studies had been performed to eliminate disadvantage of natural lectins in drug delivery that associated with the relative large size by engineering lectins with low molecular weight which characterized by nonimmunogenicity, high solubility and enhanced affinity binding to the drug, making 8

them a potent bioadhesion molecules that could bind directly the targeted cells (Lehr et al, 2004), reversibly many carbohydrate bearing drug formulation had been synthesized to target the endogenous lectins that expressed on the epithelial cell surface (Bies et al, 2003), usually lectins are expressed exclusively in certain time, certain tissues and under certain stimulation such as selectin, which is targeted by an anti-inflammatory drugs and used to deliver the drug to the site of inflammation (Ehrharadt et al, 2004). Lectins were proposed as an alternative to viral gene delivery in transferring genes (Lehr et al, 2004). Lectins themselves were used as a therapeutic agent, in treating cancer for example, by triggering apoptosis through different pathways such as mitochondria- mediated apoptosis pathway

which

characterized

by

mitochondrial

clustering,

collapsing

transmembrane potential, releasing chytochrome c and activation of caspase signal transduction pathway (Liu et al, 2010). Also lectins could trigger cytotoxicity and inhibit cancer cell growth by acting as an antiproliferative agents (De Mejia et al, 2005). Within this year a research conducted on banana lectin denoted as BanLec shows their ability to play a major role in preventing the entry of HIV-1 to lymphocytes by binding the carbohydrate of the viral envelope glycoprotein blocking it from binding its cell receptor, with strength equal to used drugs (Swanson et al, 2010). Many other lectins such as PHA of red kidney bean and samta tomato (STL) have HIV-1 reverse transcriptase inhibitor activity (Wang et al, 2006). Many clinical laboratories used plant lectins in bone marrow transplantation, cell separation and stimulation of lymphocytes proliferation and growth, the most common used mitogens are ConA and PHA, which stimulate lymphocytes to produce cytokines such as interleukin-2 (IL-2), mitogenic lectins employed to assess the immunocompetence, immunosuppressive patients with different diseases and also to evaluate the immunotherapeutic treatments (Sharon, 2007). Thus, 9

purification and characterization of lectins from a variety of plant species interest researchers in the fields of glycobiology and biotechnology. The more are known about the lectins, the wider the applications of this type of proteins can be achieved.

1.2.5 Tamarindus indica the medicinal plant: Tamarindus indica the tropical plant which had been used extensively in traditional medicine is originated in Africa where dyer savannas are found including Sudan, and later introduced to India, now it is cultivated in most all tropical countries, the monotypic genus Tamarindus belongs to legumes of the family Fabaceae. Almost all plant parts (leaves, fruits and seeds) have been widely used traditionally and this uses proved by modern medicinal science (Muthu et al, 2005) the medicinal value of this plant depends on the presence of certain phytochemical substances that poses a distinct physiological role on human body. Most of researches that had been conducted on tamarind were to extract and characterize many of the phytochemical components such as alkaloids, glycosids, phenolics, saponins, quinins, lectins and many other polypeptides (Putheti et al, 2008). It had been proved that fruits, leaves and seeds extract are possessing cooling, carminative, digestive, laxative, antiscorbutic, interferon stimulatory, immunomodularity, untiulcergenic, hepatoprotective, antibilious and antibacterial activity (Muthu et al, 2005). Methanolic extract from leaf proved in 2005 by Muthu and his colleague to play a role by affecting growth of clinical isolates of Burkhoderia pesudomallei. Seeds coat phytochemical extract had a bacteriostatic effect on Staphylococcus aureus, Salmonella typhimurium and Pesudomonus aeruginosa (Waghmare et al, 2010), but earlier in 2001, a galactose specific lectin extracted from fruit pulp that could 10

agglutinate the pathogenic strain of Echerichia coli (0157:H7) gave a postulation that tamarind had a gastrointestinal activity against bacterial infection (Rodriguez et al, 2000), moreover a novel delivery system extracted from seeds that composed from mucoadhesive polysaccharide had been used to administrate ocular antibiotics (Ghelardi et al, 2000 and 2004).

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CHAPTER TWO: MATERIALS AND METHODS 2.1 Tamarindus indica seeds collection: Season fresh tamarind fruits were brought from the local market.

2.2

Preparation of defatted acetone dried powder:

114 g seeds were ground to fine powder using coffee grinder; powder was mixed with butanol-1 (1g: 5mL) and stirred continuously for 4hrs at 4oC. The soluble protein in the resulted homogenate was precipitated with chilled acetone, then filtered with filter paper (Wattman No. 1) and washed with several folds of chilled acetone and left to dry at room temperature. The dry weight was reported. The resulted air dried preparation is called Acetone dried powder. (Konozy et al, 2002).

2.3

Preparation of crude protein extract:

Approx 437mL of physiological saline NaCl (0.145M) were added to approx 87g of fat and pigment free tamarind powder and left for 4hrs under continuous stirring at 4oC. The obtained homogenate was filtered through clean cheesecloth followed by Wattman No. 1 filter paper. Supernatant was subjected to centrifugation at 60000 r.p.m for 15mins. Obtained clear supernatant was collected; volume was recorded and preserved at -20oC till further use. This fraction is denoted here forth as Fraction A.

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2.4

Ammonium Sulfate fractionation and protein dialysis:

Protein on saline extract was fractionated with saturation with ammonium sulfate 0-30%, 30-60% and 60-80% respectively and allowed to stand at 4oC overnight. Precipitant proteins were collected by centrifugation at 60000rpm for 10minutes, dissolved in minimal volume of physiological saline (0.145M), and dialyzed against amble amount of physiological saline till free of ammonium sulfate. Table 1: table shows the amount of (NH4)2SO4 saturation added to each fraction, calculation of amount of ammonium sulfate added was done using the online calculator software from EnCor Biotechnology Inc. (www.encorbio.com/protocols/AM-SO4.htm) solution

Starting Vol.

Starting saturation Desired saturation Final Vol Added (NH4)2SO4 (%)

(%)

(ml)

(g)

(ml) Frac30

250

0.0

30

272.54

42.44

Frac60

270

30

60

296.75

50.38

Frac80

290

60

80

310.51

35.63

Before use, dialysis tubes were processed by boiling them in 100mM EDTA for 20mins. The three protein fractions obtained from ammonium sulfate fractionation were dialyzed overnight against physiological saline (0.145M) at 4oC with twice buffer changes.

13

2.5

Protein and carbohydrate estimation:

Protein concentration was estimated either by direct recording of OD280nm using bovine serum albumin (BSA) as the standard (Appendix no. 2.1.a), or by Lowry method (Lowry et al, 1951) using bovine serum albumin (BSA) as the standard (Appendix no. 2.1. c). While neutral carbohydrate contents of the protein extract was measured by phenol-sulfuric acid method as described by Dubois et al, 1956, using D-glucose as the standard (Appendix no. 2.1. b).

2.6

Hemagglutination assay:

Fresh rabbit blood cells and human erythrocytes of all blood types were collected from healthy volunteers and used in the initial hemagglutination assay. For the subsequent assays human erythrocytes of O+ type were employed. 2ml of blood were incubated with equal volume of 50mM EDTA prepared in physiological saline for 15 minutes at room temperature, and then the platelet rich plasma and puffy coat were separated by centrifugation (2000r.p.m. for 15 minutes). RBCs washed with 5mL NaCl (0.145M) several times. 4% of RBCs saline suspension was prepared prior to hemagglutination assay in U-shaped micro-titter plates. Two fold serial dilutions of the 50µL crude (7.15µg) and fractions extract in physiological saline (0.145M NaCl) were performed, and incubated with 50µL of fresh RBCs suspension for 1h at room temperature. When the erythrocytes had fully

sediment,

each

well

was

examined

for

the

agglutination.

The

hemagglutination titter was defined as the reciprocal of the least dilution that was able to induce visible erythrocyte agglutination.

14

2.7

Inhibition of hemagglutination assay:

The inhibition assays were performed in similar manner to the above hemagglutination assays. 50mM of the following carbohydrates which are all of Dconfiguration were used as a competitive inhibitors: N-acetyl-glucosamine (GluNAc), N-acetly-galactosamine (GalNAc), D-glucose, D-galactose, Dmannose, D-lactose, D-maltose, D-arabinose and D-dextrose. The possible inhibitory effect of these sugars was measured by mixing serial dilutions of the inhibitors with three hemagglutination units of the lectins followed by 2h incubation period at room temperature, before addition of equal volume of erythrocytes suspension.

2.8

EMtLs purification:

2.8.1 Affinity chromatography: Sephadex G-150 (2×8cm) column pre-equilibrated with physiological saline (0.145M) was used as an affinity column to purify both EMtL3 and EMtL6. Frac3 and Frac6 were loaded to the column separately and respectively, the column was saturated with each extract then washed with physiological saline till OD280 ≤0.02, the bounded lectins were eluted by 100mM glucose. Lectin rich fractions pooled and concentrated using Millipore centrifugation devise (cut off 10kDa) then dialyzed against physiological saline. 2.8.2 Gel filtration: Gel filtration method was used to fractionate proteins in each fraction (Appendix no. 3.1) and to determine the native molecular weight of lectin. Sephadex G-100 (Pharmacia, Sweden) column (1.5x70cm) was calibrated using gel filtration standard protein markers using Sigma Alderich MWGF70-IKT 098K6082 Kit 15

(Blue dextran 2000kDa, Albumin 66kDa, Carbonic anhydrase 30kDa, Cytochrome C 12kDa and Aprotinin 6.5kDa) (Appendix no. 2.1. d) and equilibrated with 0.145M NaCl. Fractions of 3mL were collected at a flow rate 1mL/min, fractions rich in lectin activity were pooled and concentrated using Millipore concentrating tube. 2.8.3 Gel electrophoresis: 2.8.3.1

SDS-PAGE:

Proteins were precipitated by TCA- acetone precipitation protocol (Appendix no. 1.1. d). Sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed according to Laemmli’s protocol (1970), 12.5% resolving gel of pH8.8 and 5% stacking gel (pH6.8) were prepared using 30% acrylamide. The protein bands were visualized using Coomassie Brilliant Blue and silver nitrate stain described by Blum et al, in 1987, (Appendix no. 1.1. a, c). 2.8.3.2 PAGE: Native ployacrylamide gel electrophoresis were carried according to protocol described by Williams and Reisfeld in 1964, 10% resolving acidic-PAGE of pH 4.5 were used to detect both purified EMtL3 and EMtL6 and 10% resolving basicPAGE of pH 8.6 were conducted with semi-purified EMtL8 (Appendix no. 1.1. b).

16

2.9

Lectins biophysiochemical characterization:

Lectins were characterized and assayed as described by Konozy et al (2002). 2.9.1 Effect of pH on lectins activity: 25µL of Lectins aliquots were incubated with equal volume of 20mM of different pHs ranging from (2-13) for 2h. pH was adjusted to 7.0 using 50mM NaOH or HCl. 4% erythrocytes used to assay the activity of each lectin. 2.9.2 Effect of denaturing agent on lectins stability: Varying concentrations of urea (1, 2, 3, 4, 5 and 6M) were used to study effect of denaturing agents on lectins activity by incubating 25µL of urea with equal volume of lectins aliquots at room temperature for 2h, then the residual lectin activity were assayed with 4% RBCs.

2.9.3 Effect of EDTA and metal ions on lectins activity: 300µL of each lectin were incubated overnight with 100mM of EDTA prepared in physiological saline, then dialyzed against ample of physiological saline prepared in deionized water. Hemagglutination activity was assayed before and after EDTA incubation. Sample without EDTA treatment was considered as 100% activity control. 25µL of 50mM divalent metal ions (Ca+2, Mg+2, Mn+2, Zn+2, Fe+2 and Hg+2) prepared in physiological saline were incubated with equal volume of lectin aliquots for 2h and lectins activity was determined.

17

2.9.4 Effect of Lectins on bacterial activity: Five bacterial species of both gram positive and gram negative (Echerichia coli, Salmonella typhimurium, Shigella dysenteriae, Listeria monocytogenes and Staphylococcus aureus) were cultured in nutrient broth agar plates prior to be assayed. 2.9.4.1

Effect of lectins on bacterial growth:

All the steps were carried out under aseptic conditions. Molten agar was poured in sterile Petri dishes and allowed to solidify in room temperature. Evaluation of effect of lectin on bacterial growth was performed in triplicate. Each set comprised of a negative control where respective bacterial strain is allowed to grow on agar in the absence of lectin, the test plate was composed of three tiny 0.5cm circular filter papers that were saturated with lectin. These filter papers are then carefully placed in a triangular shape on the top of the agar Petri dish. Approx 20µL bacterial strain was carefully dropped on the filter papers and incubated overnight. In the last agar Petri dish, lectin that was previously incubated with haptenic sugar was carefully added on the filter paper that was previously saturated with bacterial strain. 2.9.4.2

Bacterial agglutination assay:

Bacterial agglutination was performed in the same manner of hemagglutination. Bacteria were 2 fold serially diluted to which 3 unit of each lectin was added under aseptic conditions. Mixture was incubated for 30min and bacteria agglutination was monitored spectroscopically. The assay was repeated after incubating lectins with its competitive inhibitory carbohydrate.

18

CHAPTER THREE: RESULTS 3.1 Protein extraction and fractionation: Purification of lectin from seeds of medicinal plant Tamarindus indica started with grinding 114g seeds to fine powder followed by de-fatting and de-pigmentation by n-butanol. Extraction of the previously stated acetone dried powder (see experimental section) with physiological saline followed by fractionation with varying concentrations of ammonium sulfate which resulted in variable amount of protein yield dependant on (NH4)2SO4 saturation. Maximum amount of precipitated protein was reported with 80% (NH4)2SO4 saturation followed by 60% and finally 30% saturation. There was dramatic loss in protein content before and after dialysis, major loss of protein was exhibited by fraction 30% (Table 3). On performing the hemagglutination test of Frac3D, Frac6D and Frac8D strong hemagglutinating activity was evident for both Frac60 and 80% of (NH4)2SO4 saturation, whereas less lectin activity was detected in Frac30%. Presence of lectin in the three Frac30, Frac60 and 80% directed us to go for further fractionation. On applying Frac3D, Frac6D and Frac8D on gel filtration on Sephadex G-100, entirely different protein elution profiles were obtained (Appendix no. 3.1), and the elution profile was not always in accordance with protein peaks. Upon analyzing Frac(060%)D for lectin activity, two major activities were detected, exactly the same interesting results were obtained when Frac80D was analyzed by gel filtration, however, with different molecular weights for lectins as will come later (Fig 5 and 6). For further analysis we used gel filtration and affinity purified pooled lectin fractions.

19

3.2 Hemagglutination assay: a Hemagglutination assay of the three lectins showed that EMtL3 is AB blood type specific, EMtL6 is (A) antigen specific while EMtL8 is rabbit erythrocyte specific and do not interact with any blood group of human erythrocytes. Agglutination of RBCs by lectinn was remarkably enhanced after treating erythrocyte’s suspension with trypsin.

A

B

C

Figure 1: Microscopic view of Hemagglutination of human O+ type. (A) and (B) represent a typical erythrocytes agglutination of both EMtL3 and EMtL6 respectively, while (C) the control where RBCs sediment in the bottom of the titer plate.

20

Lectin activity (unit)

2500

2048

2000 1500

EMtL3 1000

EMtL6

512

EMtL8

500 0 A

B

AB

O

4% Human blood ABO type

Figure 2:: lectin specificity toward human erythrocyte suspension. suspension Both EMtL3 and EMtL6 are human RBCs specific while EMtL8 shows no interaction with all human erythrocytes suspension.

21

Lectin activity (unit)

4500

4096

4000 3500 3000 2500 2000

EMtL3

1500

EMtL6

1000

EMtL8

500 0

A

B

AB

O

4% Human ABO blood groups treated with trypsin

Figure 3:: Lectin activity determined by using trypsin treated RBCs (250ug/ml in 100ml Tris-base pH8.0).

22

4096

4500

Lectins activity (unit)

4000 3500 3000 2500

Rab. Untrypsinated Rab. Trypsinated

2048

2000 1500 1000 500 0

EMtL3 EMtL6 EMtL8 4% Rabbit erythrocytes Figure 4: The activity unitss of the three lectins after incubation with trypsin treated and untreated rabbit erythrocyte suspension. *Rab. Rab. untrypsinated: 4% rabbit erythrocyte suspension. *Rab. Trypsinated: 4% rabbit trypsin treated RBCs suspension.

23

Step

FracA* Frac3 D* Frac6 D Frac8 D EMtL3 G* EMtL6 G EMtL8 G EMtL3* EMtL6

Protein conc. (mg/ml)

Lectin activity unit

Lectin specific activity (unit/mg)

A

B

O

AB Rab*

1.43 2.85

32 128

64 64

16 64

64 512

8.54

2048 512

512

512 2048 239.8 59.9 59.9

0.0

0.0

6.43

0.0

0.0

512 128

512

A

B

22.3 43.4

O

AB

Rab

A

B

O

AB Rab

358 44.9

1.0 2.0

1.0 0.48

1.0 2.0

1.0 1.0 3.9 0.13

59.9

239.8

10.7

1.3

5.3

1.3 0.67

0.0

79.9

0.0

0.0

0.0

0.0 0.22

44.8 11.2 44.8 21.7 21.7 173.6

0.0

0.0

0.0

Purification (fold)

0.08

8

100

8.9

0.16

16

6.25

0.56

0.223 0.37 1.92

256

1147.9

128 1029

345 535

3.2 30.8 47.8

Table 2: Purification summary of the lectins from Tamarindus indica seeds FracA*= butanol-defatted crude saline extract. Frac3 D*= dialyzed protein. EMtL3 G*= pooled fractions that contains EMtL3 and concentrated after gel filtration on Sephadex G-100. EMtL3*= affinity purified lectins on Sephadex G-150. The colored boxes represents lectins specificity, yellow=EMtL6 A blood specific, green= EMtL3 AB blood specific and blue= EMtL8 rabbit blood specific (Rab*). Gray blocks*= represents the unperformed data.

24

Step

Total vol. (ml)

Protein conc. (mg/ml)

Protein total conc. (mg)

Carbs. conc. mg/ml

Carbs. total conc.

FracA* Frac3 S* Frac6 S Frac8 S Frac3 N* Frac6 N Frac8 N Frac3 D* Frac6 D Frac8 D EMtL3 G* EMtL6 G EMtL8 G EMtL3* EMtL6

250.0 270.0 290.0 310.5 5.5 10.5 13.5 3.0 9.5 13.0 2.0

1.43 0.99 1.026 0.65 2.95 11.72 7.32 2.85 8.54 6.43 0.08

357.5 267.3 297.5 201.8 16.2 123.1 98.8 8.55 81.1 83.6 0.16

0.395 0.253 0.211 0.187 0.179 0.283 0.091 0.038 0.057 0.041 NP*

98.8 68.3 61.2 58.1 0.985 2.972 1.223 0.114 0.542 0.533 NP

2.0 2.0 3.0 3.0

0.16 0.223 0.37 1.92

0.32 0.446 1.11 5.76

NP NP NP NP

NP NP NP NP

Table 3: Estimation of proteins and carbohydrates content during the different purification steps. FracA*= butanol-defatted crude saline extract. Frac3 S*= Supernatant after precipitating proteins of 0-30% Ammonium Sulfate saturation. Frac3 N*= Protein salted out by 0-30% (NH4)2SO4 saturation. Frac3 D*= dialyzed protein. EMtL3 G*= pooled fractions that contains EMtL3 and concentrated after gel filtration on Sephadex G-100. EMtL3*= affinity purified lectins on Sephadex G-150. NP*= Not Performed.

25

3.3 Inhibition of hemagglutination assay: It had been found that EMtL3 is completely inhibited with D-glucose (0.045mM) followed by galactose and amine sugars, the same was reported for EMtL6 (glucose and galactose specific), but it was inhibited with higher concentrations of sugars. EMtL8 has unusual sugar specificity, in which it was inhibited by maltose and mannose (Table 3). MIC* (mM) Carbohydrate

EMtL3

EMtL6

EMtL8

N-acetylgalactosamine

12.5

NI

25

N-acetylglucosamine

0.045

12.5

NI

Galactose

0.09

12.5

25

Glucose

0.045

12.5

NI

Lactose

6.25

25

25

Mannose

25

25

0.78

Arabinose

NI**

NI

NI

Maltose

NI

NI

0.39

Sucrose

NI

NI

NI

Table4: Shows the minimum inhibitory concentration of each carbohydrate needed to inhibit lectin activity. *MIC = Minimum Inhibitory Concentration needed to complete inhibitory of lectin activity ** NI= Non Inhibitory.

26

3.4 Determination of molecular weights by gel filtration: When extract that contained both EMtL3 and EMtL6 i.e., [Frac.0-60% (NH4)2SO4] was loaded to the calibrated column, two hemagglutinating proteins with molecular masses 130kDa and 11.27kDa had been detected (figure 5). Upon loading Frac8 on the same calibrated column and subsequent detection of lectin activity in fractions, two lectin activities were apparent of molecular weight 130kDa and 33.02kDa at pH7.0 (figure 6), changing the loading pH condition from pH7.0 to pH5.0 single lectin activity was found around 130kDa (Figure 7).

1.2

Protein Conc. (mg/ml)

30

Conc. Specific activity

25

1

20

0.8 15 0.6 10

0.4 0.2

5

0

0 29

49

69

89

109

Lectin Specific Activity (unit/mg)

1.4

129

Elution Volume (ml) Figure 5: Gel filtration of crude extract, fractionated by (NH4)2SO4 (0-60%) saturation. The blue primary line represents protein concentration (mg/mL) that had been eluted with physiological saline (0.145M) of 3mL intervals, while the secondary line (red) represents lectin specific activity of both EMtL3 and EMtL6. Native molecular weight had been determined with calibrated Sephadex G-100 column (1.5×70cm).

27

Protein Conc.

6

30

5

25

4

20

3

15

2

10

1

5

0

0 30

40

50

60

70

80

90

Lectin Specific Activity (unit/mg)

Protein Conc. (mg/ml)

Specific Activity

100

Elution Volume (ml)

Figure 6: Gel filtration of fraction 80%, protein at pH7.0 7.2mg protein was loaded. Fractions of 3mL were collected at a flow rate of 1mL/min. Fractions were monitored for protein concentration as well as lectin activity. Solid continuous line (black) represents the protein concentration, while solid one with circles represents EMtL8 activity detected in protein fractions.

28

250

3

Protein Conc. (mg/ml)

2.5

Specific activity

200

2 150 1.5 100 1 50

0.5

Lectin Specific Activity (unit/mg)

Conc.

0

0 20

25

30

35

40

45

50

55

60

Elution Volume (ml)

Figure 7: Gel filtration of Fraction 80% at pH 5.0. 9mg protein was loaded. Fractions of 3mL were collected at a flow rate of 1mL/min. Fractions were monitored for protein concentration as well as lectin activity.

29

3.5 Determination of protein subunits using SDS-PAGE: SDS-PAGE were used to access protein subunits for purified EMtL3 and EMtL6, two bands had been reported for EMtL3 and three subunits two of them of molecular weight higher than 100kDa were reported for EMtL6. EMtL3

EMtL6

116 kDa

66.2 kDa

45 kDa 35 kDa

25 kDa

18.4 kDa Figure 8: SDS-PAGE of purified lectins. 10µg proteins were loaded and gel was stained with Coomassie Brilliant Blue as described in materials and methods.

30

3.6 Effect of pH on Lectins activity: Both EMtL3 and EMtL6 had optimum pH on alkaline side above pH 7 Figure 9.

140 120

Lectin Activity (%)

100 80 EMtL3

60

EMtL6 40 20 0 0

2

4

6

8

10

12

14

pH

Figure 9: The effect of pH on Lectin activity of EMtL3 and EMtL6 Lectins were incubated for 2h at varying buffers ranging from pH2 –pH 13 at RT. Lectin sample was neutralized either with 0.1M NaCl or 0.1M HCl and immediately lectin activity was assayed.

31

3.7 Effect of denaturing agent on lectins activity: Incubation of purified lectins with urea of different concentrations, starting from 1M to 6M showed remarkable decreasing on lectin activity with increasing urea concentrations. EMtL3 showed less stability against denaturing than EMtL6 which is more stable in concentration ranged from 3M to 6M (Figure 10).

120

Residual Lectins Activity (%)

100 80 60 EMtL3 EMtL6

40 20 0 1

2

3

4

5

6

7

Urea (M)

Figure 10: Effect of the Denaturing agent urea on EMtL3 and EMtL6 activity Lectins were incubated for 2h with different concentrations of urea (1-6M), residual activity was calculated. Lectin in absence of urea corresponded to 100% control activity.

32

3.8 Effect of EDTA DTA and metal ions on lectins activity: EMtL6 activity was totally abolished upon incubation with chelating agent (EDTA), the activity was regained after incubating protein with 50mM of metal ions. 5 folds increase was evident upon addition of Zn+2. EDTA had no effect on EMtL3 activity however activity was increased 3 times with Ca+2, Mg+2, Mn+2 and Zn+2 (Figure 11).

200 EMtL3

180

EMtL6

160

Lectin activity (%)

140 120 100 80 60 40 20 0 Ca+2

Mg+2

Mn+2

Fe+2

Hg+2

Zn+2

Metal ions (50mM)

Figure 11:: Effect of metal ions (50mM) on EMtL3 and EMtL6 activity.

33

3.9 Bacterial inhibition and agglutination assay: EMtL3, EMtL6 and EMtL8 did not inhibit on agar-plate plate bacterial growth of (Echerichia Echerichia coli, Salmonella typhimurium, Shigella dysenteriae, dysenteriae Listeria monocytogenes and Staphylococcus aureus) aureus) at any concentration. However, on performing the agglutination test followed by microscopic studies, the three isolectins were found to agglutinate all human non-pathogenic non E. coli Figure 9. On the other hand, interestingly, EMtL6 agglutinated S. typhimurium and S. aureus. No agglutination occurred when lectins incubated with Listeria monocytogenes and Staphylococcus aureus.

A

B

Figure 12:: Microscopic view of a typical Bacterial agglutination, (A) agglutination of E. coli by the three isolectins, (B) Control (bacteria +normal saline)

34

Chapter four: Discussion and conclusion 4.1 Discussion: Genus Tamarindus is monotypic which belongs to family Fabaceae, the legume family, the one that assumed to be the third largest plant family on earth. Huge numbers of its members where suitable candidates in lectin researches, many of lectins isolated from this plant group were subjected to extensive studies where the three-dimensional structure of them had been revealed and their possible functions postulated. Among them ConA which belongs to the species Canavalia ensiformis was the most extensively studied lectin (Van Damme et al, 2008; Edelman et al, 1972). Beside the total proteins and lipids contents which had been evaluated to be about 15%, Glew and his colleagues in 2005, reported that tamarind seed’s extract contains polysaccharide units of main chain β (1, 4) glucose polymers attached to xylose α (1, 6) and galactose β(1, 2) constituents. Due to the high percentage of lipid in seeds the process of de-fatting and de-pigmentation followed by dialysis were critical during lectin purification, and in order to remove any access of free soluble carbohydrate that may likely to interfere during activity assay. The quantity of lectin in plant tissues varies dramatically. These variations may include molecular weights (Yeasmin et al, 2001), number of lectins present and sugar specificity. The fact that many lectins, which share similar or different tissue within the same plant, may exhibit similar sugar specificity and/ or molecular weights is not ruled out (Kaur et al, 2005 and Konozy et al, 2003). Gaidamashivli and his colleagues, 2004 in their work with Dioscorea batatas reported purification of two lectins of unusual sugar specificity and varying molecular weights (mannose- specific lectin, 20kDa and maltose- specific lectin, 120kDa). In compression, tamarind exhibits different numbers of lectins according to the tissue localization, in which one lectin was reported in fruit pulp (Rodriguez et al, 35

2000). Although Grant and his colleagues, in 1991 had reviewed that tamarind seeds contains only one lectin which is only capable of agglutinating rat RBCs, our investigation showed that tamarind seed contains about three lectins that are not only variable in molecular weights but also, for some of them, with dramatic variability in sugar specificity. Interestingly, tamarind lectins were extremely mosaic in many aspects. Subjecting tamarind seeds protein extracts to different concentrations of (NH4)2SO4 and subsequent assay for hemagglutinating activity in the precipitated protein, resulted in detection of three lectins i.e. [EMtL3 (0-30%), EMtL6 (30-60%) and EMtL8 (60-80%)] but, when subjecting the fruit pulp saline extract to the same successive salting out process single resulted in a single lectin which was restricted to fraction obtained upon 30% (NH4)2SO4 (data not shown). On the other hand, those three lectins of different sugar specificity, the two EMtL3 and EMtL6 are glucose and NAc-Glu sugar specific while; the fruit pulp tamarind lectin is galactose specific as reported by Rodriguez et al, 2000. Lectins with carbohydrate specificity to glucose, galactose, mannose and their derivatives are very common (Sharon, 2007; Konozy et al, 2002 and 2003). Fascinatingly, EMtL8 shows a rare specificity when inhibited by maltose; similar haptenic sugar specificity to maltose was reported by Peumans et al, 1997 and Gaidamashvili et al, 2004. In addition, the differential interactions and specificity of EMtL3, 6 and 8 with all human blood type and rabbit RBCs may again provide a clue for the mosaic property of these proteins. Although the three isolectins agglutinate rabbit erythrocytes with slight differences among them, we found that EMtL3 and EMtL6 agglutinated also human erythrocytes with specificity toward AB and A blood type respectively. Despite some studies confirmed the ability of some lectins to discriminate between different human blood antigens (Morgan et al, 2000), however this phenomena remains still quite rare in plant lectin. Boyd tested 36

plants seeds extracts for erythrocytes specificity, these studies later provided the bases for distinguishing the ABO antigen and Lewis system and identified the carbohydrates of each cell type (Boyd, 1963). The variable hapten sugar specificity of EMtL3 and EMtL6 suggested that the orientation of the hydroxyl group of the axial C4 of the hexose sugar is the important site for lectin-sugar interaction (Table 4) These findings in conjugation with the fact that AB and A blood antigens have a free hydroxyl group in C4 of glucose and NAc-Glu may favor interaction with EMtL3 and EMtL6 rather than EMtL8, which seems to possess an extended affinity side with ability to accommodate the two α (1, 4) linked glucose residues of maltose. Using gel filtration for protein fractionation was very vital step in course of lectins purification. At the same time it was useful to determine native molecular mass of isolectins. Interestingly, the finding that EMtL3 and EMtL6 are of glucose specificity was accidental. Loading of Frac30 and Frac60 on Sephadex G-100 (polysaccharide made of many glucose residues) and subsequent elution followed by lectin activity assessment were always faced by the problem of the lower eluted lectin activity as compared to total loaded activity. Elution of Sephadex G100 column with glucose resulted in high lectin activity, indicative of a glucose specific lectin. Upon saturation of Sephadex column with lectin (Frac0-60%), we could only be able to estimate molecular weight of two lectins that were eluted at variable elution volumes which were correspond to 130 and 11.27 kDa. Using of Sephadex as affinity matrix for purification of glucose specific lectins is common in literature (Samal et al, 1999; Correia et al, 1995; Kalsi et al, 1992 and Biswas et al, 2009). Fractionation of Frac80 provided also two fractions positive for lectins of molecular weight 130 kDa and 33.02kDa at natural pH, changing pH to acidic; changed the behavior of lectin and one fraction of molecular weight 130 kDa were reported, this suggested that this lectin could be found in an equilibrium 37

between the two units of 33.02 and 130 kDa at the natural condition but when the pH decreased to pH5.0 the lectins units aggregates to from a complex of the higher molecular weight. Most of the lectins purified from members of the same subfamily of tamarind, like lectins of Cassia fistula showed lectin ranging in molecular weights from 37 to 46 kDa (Ali et al, 2004). Our data concerning stability of lectins with denaturing agent (urea) were in accordance with the one published by Ali et al, who studied the effect of physical and chemical agents on stability of Cassia fistula lectins (CSLs). In their study, CSLs lost more than 70-80% of activity when incubated with 4-6M urea. However, comparatively to CSLs, EMtL3 and EMtL6 showed higher stability when treated with 3- 6M urea. Upon treatment of EMtL6 with chelating agents EDTA, lectin activity was completely abolished; these results indicated that the metal ions are loosely bound to the lectin exposed to chelating agents rather than embedded, hidden from removal. Retrieval of lectin activity by addition of Zn+2 and Ca+2 indicate that this lectin is Zn+2 and Ca+2 dependant protein. Comparatively, on working with Erythrina speciosa lectin, Konozy and his colleagues showed that Eyrthina speciosa seeds lectin is Ca+2 and Mn+2 dependant lectin and that these metal ions are easily removable (Konozy et al, 2003). Some lectins which are ingested in food could act as a dietary toxin, they could cause digestion disturbance and uncomfortablility, and they could therefore, stimulate immunogenic responses. Many legume lectins assumed to be associated with irritation of digestive tract (Hamid et al, 2009). Although of the extensive research done on the antibacterial and antiparastic activity of leaves and seeds extract of tamarind, no scientific data on tamarind lectin toxicity are available. Data are already available for action of tamarind organic methanolic extracts on bacterial strains; however, there is no report to focus on the role of tamarind 38

lectins as bacteriostatic agents. In the current investigation we demonstrated that when bacteria were treated with tamarind lectins (EMtLs) they didn’t inhibit growth of bacteria under test however clear agglutination of some of them were apparent. Beside agglutination of E. coli by EMtL6, it also agglutinated both S. aureus and S. typhimurium. These results are in accordance with Rodriguez et al results when worked with fruit pulp lectin, in which they have shown agglutination gastrointestinal pathogenic E. coli (0157:H7) (Rodriguez et al, 2000).

39

4.2 Conclusion: The popularity of tamarind juice in several parts of the globe may rule out any possible toxicity of this fruit. However, since there is no data available to indicate any nutritional values for seeds or seed extract of tamarind, though it has been shown to be used for traditional medicine (Muthu et al, 2005 and Waghmare et al, 2010), an extensive plan for toxicity studies on these lectin rich seeds became indispensable. Awaiting for the toxicity study clearance, tamarind lectins may represent a good frontier for drug and drug delivery system besides their serological biotechnology applications. The exact physiological significance of plant lectin is speculative. The apparent large numbers of tamarind lectins besides their sugar recognition complexity pattern may pave way for further investigation on possible biological role of these interesting plant proteins.

40

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characterization,

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Konozy, E.H.E., Mulary R., Faca V., Ward R.J., Greene L.J., Roque-Barreira M.C., Sabharwal S. and Bhide S.V., (2003) purification, some properties of a D-galactose binding leaf lectin from Erythrina indica and further characterization of seed lectin. Biochemie., 84: 1035-1043. Konozy, E.H.E., Bernardes E.S., Rosa C., Faca V., Greene L.J. and Ward R.J.,(2002) Isolation, purification and physiochemical characterization of a D-

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APPENDIX 1.1 Electrophoresis protocols: a. SDS-PAGE: 12.5% resolving gel were prepared as following: 4.2mL of 30% acrylamide (29.2% acrylamide and 0.8% bisacrylamide), 3.1mL of d.H2O, resolving gel (1.5M Trisbase pH 8.8) of 2.5mL and 100µL of 10% SDS were all mixed together, to initiate polymerization 10µL of TEMED and 100µL of ammonium persulphate (APS) were added. Polymerization was allowed in 1h. Then 4% stacking gel prepared by adding 1.3mL of 30% acrylamide, 4.0mL d.H2O, stacking buffer (1M of Tris-HCl pH 6.3) 2.5mL and 10% SDS 100µL, finally 10µL of TEMED and 100µL of APS were added to initiate reaction. Polymerization occurred within 1h. 10µL of sample (1mg/mL) were mixed with equal volume of sample buffer (4mL d.H2O, 0.05M Tris-OH 1mL, 0.8mL glycerol, 1.6mL of 10% SDS, 0.4mL of βmercaptoethanol and finally 0.05% of bromophenol blue 0.2mL). The run was performed in 45 minutes under 100 volt using 1L running buffer pH 8.3 of the following ingredients (Tris-OH 3.02g, glycine 14.4g, 10% SDS and 100mL of d. H2O then pH adjusted to 8.3 and volume completed to 1L). Gel then stained with Coomassie Brilliant Blue prepared by 23mL glacial acetic acid, 114mL of methanol, 0.63g of powder stain then the volume completed to 250mL) for 1h under continuous shaking, then washed several times with destaining buffer composed of 10mL glacial acetic acid, 80mL of methanol and 100mL of d.H2O until gel background became clear.

47

b. Acidic (cationic) native PAGE: 10% of resolving gel (pH 4.3) was prepared as following: 6.7mL of 1.5M acetateKOH pH 4.3 (48mL of 1M KOH, 17.2mL AcH and the volume completed to 200mL), 6ml of 50% glycerol, 30% of acrylamide (8.8mL) was added to the mixture and 4.2mL of H2O and for initiation of polymerization 40µL of TEMED and 320 µL of 10% APS were added and allowed to be polymerized for 1h. For proteins stacking 5% stacking gel was prepared by using 4mL of 0.25M acetate- KOH pH 6.8 (48mLof 1M KOH, 2.9mL AcH and the volume completed up to 200mL with d.H2O), 30% acrylamide of 1.5mL and 9.6mL of H2O were also added. Finally 15µL of TEMED and 150µL of 10% APS were used for initiation of reaction. 10µL of samples (1mg/mL) were mixed with 10µL of 1X of sample buffer which composed of 1.45mL of 50% glycerol, 0.5mL of 0.25M Acetate-KOH buffer pH6.8 and traces of methyl green tracking dye. To run the gel, 1X running buffer were used (18.7g β-alanine, 4.8mL of 0.14M acetic acid and 600mL d.H2O then pH adjusted to 4.3) under 100 volt for 2h.Gel then stained and de-stained in the same manner used with SDS-PAGE using Coomassie Brilliant Blue.

c.

Silver nitrate staining protocol:

Gel fixed with fixation buffer (50% methanol and 10% acetic acid) for 30minutes, then incubated with 5% methanol for 15minutes and allowed to be washed with H2O three times 30sec for each. Fresh cold Na2S2O3.5H2O (0.2g/L) was used to incubate gel for 120sec then re-washed with d.H2O (3× 60sec). After that a combination of (Na2CO3 3g/100mL, 37% formaldehyde 50µL/100mL and Na2S2O3.5H2O 2mL/100mL) were used as color developer for 10minutes or till bands observed. The color reaction was then stopped with 14g/L Na2EDTA and gel washed with ample amount of d.H2O. 48

d. TCA- Acetone precipitation: Reagents were mixed in a 1: 8: 1 ratio in the following order (1mL of protein sample: 8mL of 100% prechilled acetone: 1mL of 100% trichloroacetic acid (TCA) then allowed to precipitate for 1h in -20oC, after that centrifuged at (11,500 r.p.m. for 15minutes). The pellet was washed with 1mL acetone. Finally the weight of precipitated proteins was measured and then dissolved on SDS- sample buffer.

2.1 Standard Curves: a. BSA standard curve using U.V. 280nm. 0.35 0.3 O.D. (280nm)

0.25 y = 0.2659x R² = 0.96

0.2 0.15 0.1 0.05 0 0

0.5

1

1.5

BSA (mg/ml)

Figure 13: Protein calibration curve (UV 280nm). 100µg/mL working solution of bovine serum albumin (BSA) prepared from standard BSA (1mg/mL) was serially diluted and the O.D. was measured at 280nm.

49

O.D. 490nm

b. Carbohydrate standard curve:

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

y = 1.3527x R² = 0.9823

0

0.2

0.4

0.6 Glucose mg/ml

0.8

1

1.2

Figure 14: Glucose calibration curve prepared using phenol-sulfuric acid method. 1mg/mL of 100mM glucose was used as a standard solution, the phenol-sulfuric acid reaction were detected under wavelength of 490nm.

c. Protein standard curve using Lowry assay at 660nm:

O.D. (660nm)

1.2 1 0.8

y = 0.001x R² = 0.9744

0.6 0.4 0.2 0 0

200

400

600

800

1000

1200

BSA ug/ml

Figure 15: BSA calibration curve prepared using Lowry assay 1mg/mL of 100mM glucose was used as a standard solution, the reaction were detected under wavelength of 660nm.

50

d. Standard Curve for gel filtration:

6 Log Mr.

5

y = -0.6197x + 5.734 R² = 0.9743

4 3 2 1 0 0

1

2

3

4

Ve/Vo

Figure 16: Gel filtration molecular markers standard curve Logarithm molecular marker were plotted against the ratio of elution volume of the highest peaks and the void volume, proteins were estimated under wavelength of 280nm.

Gel filtration protein fractionation and profiling: Protein Concentration (mg/ml)

3.1

2.5 2 1.5 1 0.5 0 0

100 200 Elution Volume (ml)

300

400

Figure 17: Gel filtration profile of Frac30 4.5mg of protein precipitant on (NH4)2SO4 of 0-30% saturation of T.indica were allowed for fractionation using Sephadex G-100 (1.5×70cm) as described under materials and methods chapter. Proteins were eluted with 0.145M of physiological saline pH7.0. Fractions were monitored for protein at 280nm

51

Protein Concentration (mg/ml)

4 3.5 3 2.5 2 1.5 1 0.5 0 0

100

200

300

400

Elution Volume (ml)

Figure 18: Gel filtration profile of Frac60

Protein Concentration (mg/ml)

7.4mg of Frac6D were fractionated using Sephadex G-100 (1.5×70cm), eluted and estimated as described previously.

25 20 15 10 5 0 0

100

200

300

400

Elution volume (ml)

Figure 19: Gel filtration profile of Frac80 7.2mg of Frac8D were subjected to fractionation using Sephadex G-100 (1.5×70cm) Eluted fractions were assayed for lectin using 2% rabbit RBCs suspension, showed two separate positive fractions for lectin presence.

52

Protein Concentration (mg/ml)

12 10 8 6 4 2 0 0

50 Elution Volume (ml)

100

150

Figure 20: Gel filtration profile of Frac80 at pH5.0 9mg Frac8D was allowed for fractionation using Sephadex G-100 (1.5×70cm).Proteins were eluted with 10mM of acetate buffer pH5.0 and one lectin peck observed.

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