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Partial Purification and Characterization of A Lectin From
Partial Purification and Characterization of A Lectin From
1
INTRODUCTION
Bioadhesive molecules.
2
OBJECTIVES
3
REVIEW OF LITERATURE
Microbial lectins:
Bacterial lectins:
– Pathogenicity
4
– Fungal lectins:
Molds
– Aspergillus fumigatus (Tronchin et al., 2002).
– A. niger, A. versicolor, A. rugulosus and A. nidulans (Singh et al., 2008).
– Ganoderma lucidum (Thakur et al., 2007).
– Cordyceps militaris (Jung et al., 2007).
– Xylaria hypoxylon (Wang et al., 2006).
– Armillaria luteo-virens (Feng et al., 2006).
– Xercomus chrysenteron (Trigueros et al., 2003).
Yeast lectins
– Kluyveromyces bulgaricus (Al-Mehmood et al., 1992).
– Sacchromyces cerevisiae (Stratford and Pearson, 1992).
– Viral lectins:
Influenza viruses (Wiley and Skehel, 1987).
– Algal lectins:
Ulva pertusa (Wang et al., 2004).
Hypnea musciformis (Nango et al., 2005).
Bryothamnion triquetrum and B. seaforthii (Ainonz et al., 1995).
5
Purification and characterization of lectins:
6
Applications:
– identifying microorganisms
– activation of lymphocytes
– indication of synthesis of specific proteins and
enzymes
– separation of cells
– mitogens for T-cells
– targetting vaccines to the epithilium and association
of vaccines with biodegradable particles
– insecticidal properties
– toxic effects on pests
7
MATERIALS AND METHODS
Composition :
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Extraction of lectin
Washed with distilled water, 0.1M phosphate buffered saline & pressed dry
Recovered mycelium homogenized in PBS in ratio 1:2 at speed for 3-5 min in an ice bath
Collection of blood:
Blood samples from human volunteers and animals were drawn in Alsever’s
solution in the ratio 1:2 and stored at 4 °C for use following 4-5 days.
9
Preparation of cell suspension:
– Preparation of erythocyte suspension
11
– Preparation of splenocyte suspension
Excised spleen of mice was teased in MEM with the help of glass slides
13
Carbohydrate inhibition assay:
20 µl of app. diluted lectin (twice the lowest concentration capable of visible agglutination)
+
equal volume of sugar
solution to be tested
Incubated for 1 h at root temperature
Stirred for 20 min after all the salt had been dissolved
Lectin titre and protein content of supernatant and dissolved pellet was quantified
15
– Dialysis
Obtained after 40% saturation of ammonium sulphate was loaded onto dialysis tubing
(10 KDa)
Dialysed against 0.1M PBS, pH 7.2 at 4 °C for 24 h with three buffer changes every 8 h
Lectin titre and protein content of the dialysed sample was quantified
Reagents
Procedure
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0.9
0.8
0.7
0.6
0.4
0.3
0.2
0.1
0
0 100 200 300 400 500 600
Bovine serum albumin (ppm)
18
RESULTS
Human
Goat Pig Rabbit Mice Rat
A B AB O
+ + + + - + + - -
19
Plate 4.1: Biological action spectrum of lectin produced by A. niger.
A, B, AB, O: Human
Rb : Rabbit
Ra : Rat
Mi : Mice
Go : Goat
Pg : Pig
C: Control
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Table 4.2: Agglutination of lectin produced by A. niger with cells.
Cells Agglutination
Escherichia coli -
Kluyveromyces marxianus -
Splenocytes -
Lymphocytes +
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300
250
200
Titre
150
100
50
0
Untreated Protease treated Neuraminidase
treated
Fig. 4.1: Effect of enzymatic treatment of erythrocytes on their agglutination by A. niger lectin.
22
140
120
100
80
Titre 60
40
20
0
Solidified Broth
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Specific sugars Non-Specific sugars
D-ribose (well 1) D-raffinose (well 3)
L-rhamnose (well 2) D-xylose (well 4)
L-fucose (well 5) D-mannose (well 6)
D-arabinose (well 7) D-galactose (well 8)
D-sucrose (well 9) D-glucose (well 10)
D-fructose (well 12) D-maltose (well 11)
D-mannitol (well 13) D-lactose (well 14)
Bovine submaxillary mucin (well 16) Inulin (well 15)
Asialofetuin (well 17) Pullulan (well 19)
Chondroitin-6-sulphate (well 18) Inositol (well
20)
D-galactosamine hydrochloride (well 25) Meso-inositol (well
21)
N-acetyl-D-galactosamine (well 29) L-arabinose (well
22)
2-deoxy-D-glucose (well 30) D-trehalose dihydrate (well 23)
Sugar MIC
D-ribose >12.5 mM
L-rhamnose >50 mM
L-fucose >25 mM
D-arabinose >25 mM
D-fructose >12.5 mM
D-mannitol >50mM
D-sucrose >12.5 mM
Melibiose >50 mM
D-galactosamine hydrochloride >31.75 µg/ml
N-acetyl-D-galactosamine >6.25 mM
2-deoxy-D-glucose >0.19 mM
Chondroitin-6-sulphate >0.12 µg/ml
Bovine submaxillary mucin >62.5 µg/ml
Porcine stomach mucin >125 µg/ml
Asialofetuin >125 µg/ml
γ – globulin >125 µg/ml
25
1200
1000
800
Titre
600
400
200
0
3 4 5 6 7 8 9 10 11
Growth (days)
26
Table 4.5: Optimization of ammonium sulphate precipitation of A.niger lectin.
27
Table 4.6: Summary of partial purification of lectin produced by A. niger.
28
600
500
400
Titre
300
200
100
0
4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
pH
29
70
60
50
40
Titre
30
20
10
0
0 15 30 45 60 75 90 105 120
Time (min)
30
600
500
400
Titre
300
200
100
0
25 30 35 40 45 50 55 60 65 70
Temperature ( o C)
31
600
500
400
Titre
300
200
100
0
0 15 30 45 60 75 90 105 120
Time (min)
30 °C 35 °C 40 °C 45 °C 50 °C 55 °C
60 °C 65 °C 70 °C
32
CONCLUSIONS
Contd .
33
Optimum concentration of ammonium sulphate - 40%
saturation.
34
35