Advances in Bioscience and Biotechnology, 2013, 4, 999-1006 ABB
http://dx.doi.org/10.4236/abb.2013.411133 Published Online November 2013 (http://www.scirp.org/journal/abb/)
Evaluation of antimicrobial activity of a lectin isolated and
purified from Indigofera heterantha
Sakeena Qadir, Ishfak Hussain Wani, Shaista Rafiq, Showkat Ahmad Ganie, Akbar Masood,
Rabia Hamid*
Department of Biochemistry, University of Kashmir, Srinagar, India
Received 13 August 2013; revised 26 September 2013; accepted 12 October 2013
Copyright © 2013 Sakeena Qadir et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Indigofera heterantha commonly called indigo bush is
a member of leguminoseae family found in the Hima-
layan region of Kashmir. A lectin has been isolated
from the seeds of Indigofera heterantha by the purifi-
cation procedure involving anion exchange chroma-
tography on DEAE-cellulose followed by gel filtra tion
chromatography on Sephadex G 100. Molecular cha-
racterization of the lectin was done by gel filtration
and SDS-PAGE. Activity of the lectin was checked by
hemagglutination assay and the sugar specificity by
sugar inhibition tests. The antimicrobial activity of
the purified lectin was carried out by Agar disc diffu-
sion using appropriate standards. On the ion ex-
change column , the boun d protein when eluted with 0
- 0.5 M NaCl gradient emerged as three peaks—peak
I, peak II and peak III out of which only peak II
showed the hemagglutinating activity. The lectin fur-
ther resolved into two peaks G1 and G2 on gel filtra-
tion, with the lectin activity residing in G1, corre-
sponding to a molecular weight of 70 KDa. The puri-
fied lectin named as Indigofera heterantha Lectin
(IHL) produced a single band on SDS PAGE (18
KDa), revealing the tetrameric nature of the lectin. It
agglutinated human erythrocytes (A, B, AB, and O).
Hemagglutination was inhibited by D-galactose, D-
mannose and D-arabinose. The lectin is reasonably
thermostable showing full activity within a tempera-
ture range of 30˚C to 90˚C. pH stability of the lectin
falls in the range of 2 - 9. IHL demonstrated a re-
markable antibacterial activity against the pathogenic
bacteria Klebsiella pneumoniae, Staphylococcus au-
reus, Escherichia coli, and Bacillus subtilis. IHL also
inhibited the growth of phytopathogenic fungi Asper-
gillus niger, Aspergillus oryzae and Fusarium oxys-
porum.
Keywords: Indigofera heterantha Lectin;
Hemagglutination; Antimicrobial
1. INTRODUCTION
Lectins are proteins of non-immune origin that bind to
carbohydrates and sugar containing substances in a spe-
cific and reversible way or precipitate glycoconjugates
[1]. The main properties of lectins are based on their
ability to interact with carbohydrates and thus combine
with glycocomponents on the cell surface, leading to
their biological properties [2]. Lectins are able to tightly
bind to and cause the precipitation of specific polysac-
charides and glycoproteins because they are polyvalent.
They vary, however, in molecular size, amino acid com-
position, metal ion requirements, and three-dimensional
structure [3]. Lectins can recognize mono-, oligo- or
polysaccharides, as well as glycoconjugates and thus
recognize glycoproteins or glycolipids, e.g. on the sur-
face of cells [4]. Generally, lectins are glycoproteins con-
sisting of subunits ranging in molecular mass from 25 to
35 kDa, arranged as dimers or tetramers, and existing as
multiple isoforms sharing similar biochemical properties
[5]. The subunits are usually identical or very similar,
made up of single polypeptide chains that are encoded by
different genes or by a family of closely related genes [6].
In addition to their carbohydrate binding activity, some
of the lectins such as those of hemagglutinin, galactosi-
dase, polynucleotide adenosine glucosidase, or ribosome
inactivating proteins, also show other biological activi-
ties [7]. Lectins in higher plants defend against patho-
genic bacteria and fungi by recognizing and immobiliz-
ing the infecting microorganisms via binding, thereby
preventing their subsequent growth and multiplication.
They act as a sort of immune system for plants by
“sticking” themselves to the structural carbohydrates
(sugars) of invaders. Recently, lectin biology has applied
these tools in the biomedical field and also in the treat-
*Corresponding author.

S. Qadir et al. / Advances in Bioscience and Biotechnology 4 (2013) 999-1006 1000
ment of diseases, including cancer [8]. Another applica-
tion reported in the literature involving lectins is their
antimicrobial activity where lectins may act against mi-
croorganisms by interfering with their growth and play-
ing a role in defense systems [9]. Antibacterial activity
on Gram-positive and Gram-negative bacteria occurs
through the interaction of lectin with components of the
bacterial cell wall including techoic and teichuronic acids,
peptidoglycans and lipopolysaccharides; study revealed
that the isolectin I from Lathyrus ochrus seeds binds to
muramic acid and muramyl dipeptide through hydrogen
bonds between ring hydroxyl oxygen atoms of sugar and
carbohydrate binding site of lectin and hydrophobic in-
teractions with the side chains of residues Tyr100 and
Trp128 of isolectin I [10]. The inhibition of fungal
growth can occur through lectin binding to hyphas re-
sulting in poor absorption of nutrients as well as by in-
terference on spore germination process [11]. The poly-
saccharide chitin is constituent of fungi cell wall and
chitin-binding lectins showing antifungal activity; im-
pairment of synthesis and/or deposition of chitin in cell
wall may be the reasons of antifungal action [12]. Proba-
bly the carbohydrate-binding property of lectin is in-
volved in the antifungal mechanisms and lectins of dif-
ferent specificities can promote distinct effects. In this
report, we describe the purification and characterization
of a lectin from Indigofera heterantha seeds and also the
evaluation of its potential antimicrobial activity.
2. MATERIALS AND METHODS
The plants (Indigofera heterantha) were collected from
the premises of the Department of Botany of Kashmir
University and were authenticated from Centre of Plant
Taxonomy of the same department. DEAE-cellulose and
Sephadex G-100 were purchased from Sigma Aldrich
Company, USA. Other chemicals were of highest purity
grade.
2.1. Isolation and Purification of Lectins from
Indigofera heterantha Seeds
Twenty five grams of dry Indigofera heterantha seeds
were allowed to swell in distilled water at 4˚C overnight.
The seeds were then ground in a mortar and pestle and
proteins extracted by suspending the ground seed meal in
0.15 M NaCl (10% w/v) for 1 hour at room temperature
and subjecting the mixture to homogenization in a Remi
auto mix blender for 10 min. The homogenate thus pro-
duced was stirred for 2 h at 4˚C and then filtered through
double layered cheesecloth. The filtrate was centrifuged
at 5000 × g for 20 min at 4˚C. The pellet was discarded.
The storage proteins present in the supernatant were pre-
cipitated by slowly adding 1 M acetic acid to the stirred
solution until pH 5.0 was reached. After an additional
hour of stirring at 4˚C, the suspension was again centri-
fuged at 5000 × g for 20 min. The pellet was again dis-
carded and the supernatant readjusted to pH 8.0 by the
addition of 0.1 M NaOH. The supernatant thus obtained
was dialyzed against 20 mM phosphate buffer, pH 7.2
for 24 hr. The viscous extract thus obtained was the
crude extract and was stored at 4˚C.
To purify the lectin further, the crude extract was sub-
jected to ion exchange chromatography on DEAE cellu-
lose column in 50 mM Tris-HCL buffer, pH 8. The pro-
tein was eluted using linear sodium chloride gradient
from 0 to 0.5 M. The active fractions were pooled and
dialyzed against Tris-HCL buffer, pH 8.
To check the size homogeneity, the active fractions
eluted from ion-exchange column were chromatographed
on Sephadex G 100 column in 0.05 M Tris-HCl, pH 8.0.
The charge homogeneity of the fractions eluted from gel
filtration column was checked by polyacrylamide gel
electrophoresis. Estimation of the subunit molecular
weight of the purified lectin was done by SDS-PAGE.
2.1.1. Pr otein Esti m a t ion
Protein concentration was determined by the method of
Lowry et al. [13] using BSA as the standard protein.
2.1.2. Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis in presence and ab-
sence of SDS was performed at pH 8.3 on 10% and 12%
gels respectively with a discontinuous buffer system. The
gels were stained with Coomassie Brilliant Blue R-250.
2.2. Hemagglutination Assay and Inhibition of
Hemagglutination
The hemagglutination activity of the lectin was deter-
mined by a slight variation of the method devised by
Peumans et al. [14], using human erythrocytes bearing
blood groups A, B, O, AB and chick erythrocytes. The
human blood was collected from SKIMS, Srinagar and
chick blood was obtained from a local veterinary outlet.
The erythrocytes were separated by centrifugation of the
blood at 2500 g (5000 rpm) for 10 min, and the R.B.Cs
washed thrice with normal saline. Finally the erythro-
cytes were suspended in normal saline to obtain a final
concentration of 3% erythrocyte suspension. The assay
of agglutination was carried out on glass slides by mix-
ing the erythrocytes with the test solution. 50 µl erythro-
cyte suspensions (A, B, O, AB and Chick erythrocytes)
were taken on a slide. To this, 50 µl of the test solution
was added. After an incubation period of 15 min at room
temperature, agglutination was monitored unaided on the
slides. Also the control slide, using the buffer instead of
the lectin solution was run simultaneously. The lectin
activity has been expressed as EU (Erythroagglutinating
Unit). One erythroagglutinating unit (EU) is defined as

S. Qadir et al. / Advances in Bioscience and Biotechnology 4 (2013) 999-1006 1001
the minimum amount of the lectin per ml required to
give positive agglutination of 1 ml of a 5% human ery-
throcyte suspension. EU is expressed as μg of lectin per
ml of the protein solution.
The carbohydrate specificity was investigated by ob-
serving the inhibition of the lectin induced hemaggluti-
nation by various sugars namely D-glucose, D-galactose,
D-mannose, fructose, lactose, maltose, sucrose, ribose
and sugar derivatives like N-acetyl galactosamine and
N-acetyl glucosamine. The inhibition assay was per-
formed on glass slides. Different dilutions of the above
sugars (final volume 20 µl) were added to glass slides on
which agglutination was performed. To each dilution, 20
µl of purified lectin was added. The mixture was incu-
bated at room temperature for 1 hr after which 80 µl of
3% suspension of erythrocytes was added to each slide.
The minimum concentrations of each sugar capable of
fully inhibiting agglutination after 1 hr at room tempera-
ture were noted.
2.3. Effect of pH and Temperature on the
Stability of the Lectin
The pH dependence of the lectin was determined by in-
cubating 50 μg of IHL with buffers in different pH: 0.1
M glycine/HCl (pH 2 - 3), 0.05 sodium acetate/acetic
acid (pH 4 - 5), 0.05 M potassium phosphate (pH 6 - 7),
0.05 M Tris-HCl (pH 8 - 9) and 0.1 M glycine-NaOH
(pH 10 - 11) for 5 hrs at 25˚C and pH adjusted to 7.2 just
prior to hemagglutination assay. The effect of tempera-
ture on the agglutinating activity of the IHL lectin was
determined by carrying out assay at different tempera-
tures according to the method described by Ngai and Ng
[15]. The purified lectin was incubated in a water bath
for 30 min at various temperatures: 10˚C, 20˚C, 30˚C,
40˚C, 50˚C, 60˚C, 70˚C, 80˚C, 90˚C and 100˚C, and then
cooled to 20˚C in each case. Hemagglutination assay was
carried out as previously described. The assay was per-
formed in duplicate [16].
2.4. Molecular Weight Determination
The subunit molecular mass of the lectin was estimated
by discontinuous SDS-PAGE according to method of
Laemmli [17]. Native molecular mass was determined by
gel filtration on Sephadex G-100 column using the fol-
lowing protein markers: BSA (66 kDa), Ovalbumin (45
kDa), Pepsin (34.7 kDA) and cytochrome c (12.4 kDa).
The gel filtration data was treated according to the rela-
tion given by Andrew [18].
2.5. Evaluation of Antimicrobial Activity
For the evaluation of antibacterial activity of Indigofera
heterantha lectin, firstly all the glassware was sterilized
in an autoclave. After sterilization, the subsequent steps
were the preparation of media, selection of the test or-
ganisms and sensitivity tests of antibacterial activity.
Purified IHL was used for analysis. Ciprofloxacin 5
µg/ml was used as standard. Human pathogenic bacteria
Pseudomonas spp. Escherichia coli, Shigella bodyi,
Klebsiella pneumonea, Streptococci, Salmonella typhi
and Staphylococcus aureus obtained from Department of
Microbiology, Sheri Kashmir Institute of Medical Sci-
ences Srinagar were used to check the antibacterial activ-
ity. Antifungal activity was checked against Aspergillus
niger, Aspergillus Oryzae and Fusarium oxysporum and
were obtained from Sheri Kashmir University of Agri-
cultural Science and Technology, Shalimar. The Agar
disc diffusion method was used to determine the anti-
microbial activity. This test was done according to the
method of Edward [19] and Lansing [20]. The microbial
(bacterial/fungal) colony was picked by the inoculating
wire and the media plates were subsequently inoculated
with specific microbial strains and labeled accordingly.
The inoculated plates were left to dry for at least 5 - 10
minutes, after which Whatman filter paper discs 6 mm
(previously saturated with crude extract and purified
lectin) were placed on the plates with the help of a steril-
ized forcep. Standard antibiotic discs of ciprofloxacin
(positive control) were also impregnated on plates at
previously labeled positions. Loaded plates were kept as
such for some time under Laminar hood and then in-
cubated at 37˚C for 24 hours in case of bacterial strains
and 48 hours in case of fungal strains in the incubator. A
transparent ring around the paper disc signified antim-
icrobial activity. Zone diameters (mm) around each of
the discs were measured to the nearest mm.
3. RESULTS
3.1. Isolation and Purification
A lectin was isolated and purified from the Indigofera
heterantha seeds by ion exchange chromatography on
DEAE-Cellulose followed by gel filtration on Sephadex
G-100. The crude extract obtained after homogenization
of the finely ground seed meal in 0.15 M NaCl was as-
sayed for hemagglutinating activity using human eryth-
rocytes of blood group types A, B, AB and O. This was
then applied on to a DEAE-Cellulose column equili-
brated with 50 mM Tris-HCl buffer, pH 8 (containing
0.02% NaN3). Major portion of the protein in crude ex-
tract passed unbound through the column. This fraction
did not carry any hemagglutinating activity. The bound
protein when eluted with 0 - 0.5 M NaCl gradient
emerged as three peaks—peak I, peak II and peak III out
of which only peak II showed the hemagglutinating ac-
tivity. Typical profile is shown in Figure 1. Fractions
under the active peak were pooled together and concen-
trated, and then subjected to gel filtration on Sephadex

S. Qadir et al. / Advances in Bioscience and Biotechnology 4 (2013) 999-1006 1002
Figure 1. Ion exchange chromatography of the crude extract on
DEAE-cellulose column (1.6 × 10 cm). About 60 mg of protein
was applied on DEAE-cellulose column pre-equilibrated with
50 mM Tris-HCL buffer, pH-8. The protein was eluted using 0
- 0.5 M NaCl gradient in 5 ml fractions at a flow rate of 30
ml/hr.
G-100 column (2.6 × 40 cm) where it resolved into two
peaks, G1 and G2 (Figure 2). Lectin activity resided
majorly in G1. The fractions in the peak (G1) were again
pooled together, concentrated and stored at 4˚C in 0.05
M Tris-HCl, pH 8.0 containing 0.02% NaN3. This prepa-
ration was called the Indigof era heterantha lectin or IHL,
and was used for physicochemical and biological char-
acterization. Data on the purification of Indigofera het-
erantha lectin is summarized in Table 1. The yield of the
lectin was about 0.46% and about a 5.2 fold purification
of the lectin was achieved.
3.2. Electrophoretic Analysis
The charge homogeneity of the lectin was established by
subjecting the lectin to polyacrylamide gel electrophore-
sis under native conditions. The electrophoretogram shows
that the lectin moved as a single band (Figure 3). In 12%
SDS-PAGE, the lectin again moved as a single band,
establishing that the lectin is composed of similar type of
subunits (Figure 4).
3.3. Characterization
3.3.1. Molecular Weight Determination
Molecular weight of the Indigofera heterantha lectin as
determined by gel filtration on a Sephadex G-200 col-
umn (2.6 × 40 cm) equilibrated in 0.05 M Tris-HCl
buffer, pH 8 was about 70 KDa (Figure 2). In SDS-
PAGE under reducing conditions, the lectin moved as a
single protein band with molecular weight corresponding
to 18 KDa. The results of SDS-PAGE (Figure 4) to-
gether with gel filtration data revealed that IHL (Indigo-
fera heterantha lectin) is a homotetramer.
3.3.2. Hemagg l uti nation
The Indigofera heterantha lectin (IHL) does not show
any marked blood group specificity (Table 2 ). As is evi-
dent, the lectin agglutinated human erythrocytes of all
blood groups, being somewhat more specific towards
blood group A erythrocytes. Extent of hemagglutination
was found to be same, when human erythrocytes of
blood group A, B, AB and O were incubated with puri-
fied IHL for overnight at 7˚C and for 6 hr at 27˚C &
37˚C.
3.3.3. Carbohydrate Specificity
In order to determine the sugar specificity of the lectin,
inhibition and reversal of inhibition by a number of sug-
ars and sugar derivatives was studied. In each case the
ability of the sugar to inhibit agglutination was measured.
Results on such specificity studies are shown in Table 3.
It is clear from the table that arabinose is the most potent
inhibitor of the Indigofera heterantha lectin mediated
hemagglutination followed by the disaccharide sucrose.
Galactose also inhibits agglutination though at very high
concentrations.
3.4. Effect of pH and Temperature
The examination of IHL activity in different pH (pH 2 -
13) values showed that the lectin was stable in the pH
range of 2 - 9 (Figure 5) indicating that the amino acid
residues involved in carbohydrate binding are not af-
fected by changes in pH in this range. The activity how-
ever falls of rapidly thereafter, with essentially all activ-
ity lost after pH 12. The purified lectin sample was found
to be heat stable up to 90˚C, at incubation temperatures
of 30 to 90˚C (Figure 6). Even heating at 100˚C for 30
minutes caused a loss of only 25% of its original activity.
However the hemagglutinating activity of the lectin was
completely lost when exposed to 110˚C.
3.5. Antimicrobial Activity
3.5.1. Antibacte ri a l Act i vity
Purified lectin obtained from ion exchange chromatog-
raphy and gel filtration chromatography as well as the
crude extract were tested against different bacterial
strains and compared to that of antibacterial antibiotic,
ciprofloxacin. The results of the sensitivity test are
shown in Figure 7. Purified IHL (500 μg/ml) exhibited a
significant antibacterial effect on four strains namely
Klebsiella pnuemoniae, Staphylococcus aureus, Escheri-
chia coli, and Bacillus subtilis. The diameters of the
zones of inhibition by the addition of IHL were 10 mm, 6
mm, 4 mm and 4 mm respectively. The diameters of the
zones of inhibition with the standard drug used were 22
mm, 20 mm, 22 mm and 13 mm for the four strains re-
spectively. Results with the crude extract were not sig-
ificant. n

S. Qadir et al. / Advances in Bioscience and Biotechnology 4 (2013) 999-1006 1003
Table 1. Purification profile of Indigofera heterantha lectin.
Vo l u m e
(ml)
Purification step Protein
(mg/ml)
Total protein
(mg)
Activity
(EU)*
Total
activity
Specific activity
(E.U*/mg)
Fold
purification
Yeild
%
Crude extract 500 9 4500 81 40,500 9 1 100
Ion exchange
Chromatography 10 1.7 17 52.02 520.2 30.6 3.4 1.28
Sephadex gel
chromatography 4 1 4 46.80 187.2 46.8 5.2 0.46
*One erythroagglutinating unit (EU) is defined as the minimum amount of the lectin per ml required to give positive agglutination of 1 ml of a 5% erythrocyte
suspension. EU is expressed in microgram of lectin/ml of the protein solution.
Table 2. Blood group specificity of purified Indigofera hete-
rantha lectin.
Blood group Specific activity (EU*/mg)
A 46.8
B 35.3
O 31.2
*One erythroagglutinating unit (EU) is defined as the minimum amount of
the lectin per ml required to give positive agglutination of 1 ml of a 5%
erythrocyte suspension. EU is expressed in μg of lectin/ml of the protein
solution.
Table 3. Carbohydrate Inhibition of Indigofera heterantha lec-
tin mediated hemagglutination. Figure 2. Gel filtration of fraction PII from ion exchange col-
umn on Sephadex G-100 column (2.6 cm × 40 cm). About 20
milligram of protein was applied on the column pre-equili-
brated with 50 mM Tris-HCL buffer, pH 8.0. The protein was
eluted using the same buffer in 3 ml fractions at a flow rate of
30 ml/hr.
Sugar Minimal inhibitory concentration (mM)*
Arabinose 0.039
Sucrose 0.188
D-galactose 0.275
Ribose No inhibition
N-acetyl galactosamine No inhibition
Raffinose No inhibition
Lactose No inhibition
Mannose No inhibition
Glucose No inhibition
*Minimum sugar concentration necessary for complete inhibition of agglu-
tination of human erythrocytes by Indigofera heterantha lectin solution of 4
µg/ml.
3.5.2. An ti fungal Ac tivity
In vitro antifungal susceptibility by IHL was determined
against three phytopathogenic fungi Aspergillus oryzie,
Aspergillus niger and Fusarium oxysporum with cipro-
floxacin as positive control. The lectin showed some,
though not significant inhibition of growth against all the
three strains with a zone inhibition diameter of 9 mm, 3
mm and 6 mm respectively in each case (Table 4). Figure 3. Polyacrylamide Gel Electrophoresis
(PAGE) pattern of Indigofera heterantha lectin
under native conditions. About 40 μg of the
purified IHL was applied on 10% polyacrylamide
gel. PAGE was performed in Tris-glycine buffer,
pH 8.0. Current applied was 25 mA for 2 hrs at
room temperature.
4. DISCUSSION
Legume lectins represent the largest and most thoroughly
studied family of the simple lectins. The members of this

S. Qadir et al. / Advances in Bioscience and Biotechnology 4 (2013) 999-1006 1004
Figure 4. SDS-Polyacrylamide gel electrophoresis
of Indigofera heterantha lectin. About 60 μg of
lectin was electrophoresed on 12% polyacrylamide
gel at a current of 3.0 mA/well in presence of 0.1%
SDS. Tris-glycine buffer pH 8.0 was used. The
staining reagent used was Coomassie brilliant blue
G-250. Lane 1: molecular weight markers (66 KDa,
45 KDa, 35 KDa, 25 KDa, 18 KDa and 14 KDa).
Lane 2: Purified Indigofera heterantha lectin.
Figure 5. Effect of pH variations on IHL activity. The pH de-
pendence of the lectin was determined by incubating 50 μg of
IHL with buffers in different pH: 0.1 M glycine/HCl (pH 2 - 3),
0.05 sodium acetate/aceticacid (pH 4 - 5), 0.05 M potassium
phosphate (pH 6 - 7), 0.05 M Tris-HCl (pH 8 - 9) and 0.1 M
glycine-NaOH (pH 10 - 11) for 5 hrs at 25˚C and pH was ad-
justed to 7.2 just prior to hemagglutination assay.
protein family consists of two or four subunits (protom-
ers), either identical or slightly different each with a sin-
gle small carbohydrate combining site with the same
specificity. A lectin was isolated from seeds of Indigo-
fera heterantha using a combination of ion exchange and
gel filtration chromatographies. The lectin is a tetramer
and exhibits a molecular weight of 70 KDa with a sub-
unit Mr of 18 KDa. The tetrameric nature of IHL is
similar to the lectin obtained from Phaseolus acutifolius
Figure 6. Effect of temperature on IHL activity. The tempera-
ture dependence of the lectin was determined by incubating the
agglutination mixture (50 μg of IHL in Tris-HCl buffer, pH 8)
at different temperatures for 30, cooled to 20˚C and determin-
ing the hemagglutination activity.
(a)
(b)
(c)
(d)
Figure 7. Antibacterial assay of Indigofera heter-
antha lectin with four different bacterial strains (a)
Klebsiella pnuemoniae; (b) Staphylococcus aureus;
(c) Escherichia coli; and (d) Bacillus subtilis. The
agar disc diffusion method was used to determine
antibacterial activity. Ciprofloxacin (5 μg/ml) was
used as standard.

S. Qadir et al. / Advances in Bioscience and Biotechnology 4 (2013) 999-1006 1005
Table 4. Antifungal activity of Indigofera heterantha lectin.
S.No Organism Diameter of zone of inhibition (mm)
Crude
(500 µg/ml)
Purified IHL
(500 µg/ml)
Ciprofloxacin*
(5 µg/ml)
1 Aspergillis
niger 5 3 27
2 Aspergillis
Oryzie 12 9 32
3 Fusarium
oxysporum 10 6 27
*Ciprofloxacin was used as a standard.
var. escumite [21]. IHL showed no specificity in its abil-
ity to hemagglutinate human (A, B, AB, and O) erythro-
cytes as the lectin from Egyptian Pisum sativum seeds
and Erythrina variegata lectin [22]. The hemagglutina-
tion assays showed that the lectin activity was inhibited
by sucrose, D-galactose and arabinose. The carbohydrate
specificity was similar to the lectin from Capsicum an-
num [23]. Thermal denaturation results of IHL showed
that the lectin is stable up to 90˚C. Only 25% hemagglu-
tinating activity was lost when heated at 100˚C for 30
minutes and was completely lost at 110˚C. The heat sta-
ble nature of IHL is similar to the protein reported by
Ngai et al. [24] and the lectin from Eugenia uniflora
seeds [9]. The loss of hemagglutinating activity with in-
creasing temperature is evidently due to heat induced
denaturation of the lectin. This denaturation may ex-
pectedly weaken the interaction between lectin and the
carbohydrate ligand leading consequently to attenuated
agglutinating activity. The studied lectin showed a re-
markable antibacterial activity against Bacillus subtilis,
Staphylococcus aureus, Escherichia coli and Klebsiella
pneumon ea, Although the mechanism of action of the
peptides has not yet been elucidated in detail, the pre-
sented data confirm the in vitro antibacterial activity of
IHL against pathogenic bacteria. It has been proposed
that the proteins with antibacterial action form a channel
on cell membrane and the cell dies as a result of the out
flowing of cellular contents, this mechanism being dif-
ferent from that of antibiotics [25]. The observed anti-
fungal activity of IHL against Aspergillis niger, Asper-
gillis Oryzie and Fusarium oxysporum agrees with the
results obtained from other plant legume lectins [26-28].
Antifungal activity has been related to the lectin carbo-
hydrate binding property, that might endow lectin mole-
cules with binding activity towards certain carbohydrate
components in the fungal cell wall affecting its activity
and viability as most lectins recognize either N-acetyl-
neuraminic acid, N-acetylglucosamine, N-acetyl-galac-
tosamine, galactose, mannose, or fucose in accordance
with the conclusion of Lis and Sharon [29]. These results
point out that future finding of lectin applications from
plants can be of great importance for clinical microbial-
ogy and possible therapeutic applications.
5. CONCLUSION
Indigofera heterantha is an indigenous plant of the Hi-
malayan region, but before this study no bioactive com-
ponent has been isolated from it. IHL represents the first
isolated proteinaceous constituent of the plant and being
active against several pathogenic microorganisms could
be used as an antimicrobial agent for animal and plant
infections.
6. ACKNOWLEDGEMENTS
The authors express gratitude to UGC, India and to the Department of
Biochemistry, University of Kashmir, Srinagar for providing financial
assistance and other necessary support.
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