Purification and characterization of three galactose specific lectins
from Mulberry seeds (
Morus
sp.)
Tanzima Yeasmin, Md Abul Kashem Tang, Abdur Razzaque and Nurul Absar
Department of Biochemistry, University of Rajshahi, Rajshahi-6205. Bangladesh
Three lectins were extracted and purified from mulberry
seeds by gel filtration of 100% ammonium sulfate saturated
crude protein extract followed by ion-exchange chromato-
graphy on DEAE and CM-cellulose. The lectins were found
to be homogeneous as judged by polyacrylamide disc gel
electrophoresis. The molecular masses of the lectins as
determined by gel filtration were 175 000 for MSL-1,
120 000 for MSL-2 and 89 500 for MSL-3. MSL-1 is dimer
in nature, with the two monomers held together by disulfide
bond(s), while MSL-2 and MSL-3 contain four nonidentical
subunits that are held together by nonionic hydrophobic
interactions.
The lectins agglutinated rat red blood cells and this
agglutination was inhibited specifically by galactose,
methyl-a-
D-galactopyranoside, methyl-b-D-galactopyrano-
side, lactose and raffinose. The lectins MSL-1, MSL-2 and
MSL-3 contained 5.7, 5.4 and 4.5% neutral sugars, respec-
tively, and the sugar composition of the lectins was glucose
and mannose for MSL-1 and galactose for both MSL-2 and
MSL-3. The lectins exhibited strong cytotoxic effect in brine
shrimp lethality bioassay.
Keywords: mulberry seeds; galactose-specific lectins;
subunit structure; hemagglutination; cytotoxicity.
Mulberry (Morus alba L.) is the sole host plant of the

MATERIALS AND METHODS
Mulberry seeds were collected from the experimental plot of
Bangladesh Sericulture Research and Training Institute,
Rajshahi. Sephadex G-150, Sephadex G-75, DEAE-
cellulose, CM-cellulose and Sepharose 4B were purchased
from Sigma Chemical Co. All the other reagents used were
of analytical grade. Unless otherwise specified, all
operations were performed at 4 8C.
Preparation of fat free dry powder
The seeds were crushed into paste using a mortar and pestle.
This was then mixed uniformly with precooled petroleum
ether in a homogenizer at 4 8C. The homogenate was filtered
through a clean muslin cloth. The process was repeated at
least twice in order to obtain lipid-free homogenate. Finally,
the filtrate was clarified further by centrifugation at 8000 g,
4 8C for 10 min The precipitate obtained was collected and
air-dried at room temperature.
Preparation of crude protein extract
The protein from fat free dry powder was extracted with five
different solvents (1% CH
3
COOH; 10 mM Tris/HCl buffer,
pH 8.4; 10 m
M phosphate buffer, pH 7.2; 20 mM acetate
buffer, pH 5.0 and distilled water). Water was used for
preparation of crude protein extract from fat free dry powder
as the highest ratio of absorbance at 280 nm and 260 nm
was found in distilled water [9]. The fat free dry powder was
mixed uniformly with precooled distilled water (4 mL
:

0.18 and 0.3
M).
CM-cellulose chromatography. The above protein fractions
needed for further purification after DEAE-cellulose
chromatography was dialyzed 12 h against distilled water
and overnight against 5 m
M phosphate buffer, pH 6.5 and
then loaded onto the column. The protein was eluted from
the column stepwisely using the same buffer containing
0.2
M NaCl.
Polyacrylamide disc gel electrophoresis. Polyacrylamide
disc gel electrophoresis was conducted at room temperature,
pH 8.4 on 7.5% gel as described by Ornstein [10] and 1%
amido black was used as staining reagent.
Characterization of lectin
Molecular mass determination: gel filtration. The molecular
masses of the lectins were determined by gel filtration on
Sephadex G-150 (0.75 Â 100 cm) using lysozyme, trypsin
inhibitor, a-amylase, BSA and b-amylase as reference
proteins.
Molecular mass determination: SDS/PAGE. SDS/PAGE was
conducted on a 10% acrylamide gel according to Weber &
Osborn [11] and the marker proteins used were same as
those used for the gel filtration. Dissociation and reduction
of proteins were performed by heating for 5 min at 100 8C
in 0.1% SDS with 0.1% 2-mercaptoethanol and the proteins
were stained with Coomassie Brilliant Blue R-250.
Hemagglutination studies
Hemagglutinating activity was assayed by the serial dilution

of Dubois et al. [15] with
D-glucose as the standard.
For identification of sugars, the lectins were hydrolyzed
with 1
M HCl for 4 h at 100 8C under vacuum. The sugar
component was determined by the one-dimensional TLC
method described by Joseph & Murrell [16] using different
standard sugars. The chromatogram was developed with the
solvent: Isopropanol, acetic acid and water (3 : 1 : 1, v/v/v)
and the spots were identified by spraying with aniline/
phthalate solution.
Toxicity study
Cytotoxicity was studied using the eggs of the brine shrimp
nauplii (Artemia salina L.). Eggs were placed in one side of
a small tank divided by a net containing 3.8% NaCl solution
for hatching. In the other side of the tank, a light source was
placed in order to attract the nauplii. Two days were allowed
for the hatching of all the eggs and sufficient maturation of
the nauplii for the experiment described by Meyer et al.
[17].
From the stock solution of the lectins (0.9 mg
:
mL
21
), 10,
20, 40, 80 and 160 mL were placed in different vials and
NaCl solution was added to each vial make the volume up to
5 mL; the final concentration of the sample in the vials
became 1.8, 3.6, 7.2, 14.4 and 28.8 mg
:

pH 8.4 at 4 8C for 24 h. After removal of the insoluble
material, the clear supernatant was applied to a DEAE-
cellulose column at 4 8C, previously equilibrated with the
same buffer, and the protein was eluted by a linear gradient
of NaCl from 0.0 to 0.3
M in the buffer. The components of
F-1 were eluted as a single, but broad, peak indicating the
presence of more than one component (data not shown). In
order to separate the components, the elution was carried out
in a stepwise fashion with an increasing concentration of
NaCl in the same buffer. Fig. 2, shows that the components
of F-1 fraction were separated into three different fractions,
F-1a, F-1b and F-1c, which were eluted with the buffer
containing 0.06, 0.18 and 0.3
M NaCl, respectively. The
fractions indicated by the solid bars were pooled separately
and their homogeneity was checked by polyacrylamide disc
gel electrophoresis. It is evident from Fig. 2 (inset) that the
fractions F-1b and F-1c contained pure protein as they gave
single bands while F-1a gave more than one band on the gel.
The fraction F-1a was further purified by CM-cellulose
chromatography (see below). All three fractions displayed
lectin activity.
The fraction F-1a obtained after DEAE-cellulose
chromatography was dialyzed overnight against 5 m
M
phosphate buffer, pH 6.5, and then applied to a CM-
cellulose column at 4 8C. Fig. 3 shows that fraction F-1a was
separated into two fractions, F-1a
0

filtration, were estimated to be 175 000, 120 000 and
89 500 Da for F-1a (mulberry seed lectin-1 i.e. MSL-1),
Fig. 1. Gel filtration of crude protein extract on Sephadex G-75.
The crude extract (85 mg) was applied to the column (2.5 Â 100 cm),
pre-equilibrated with 5 m
M phosphate buffer, pH 7.6, at 4 8C and
developed with the same buffer.
Fig. 2. Ion-exchange chromatography of
fraction F-1 on DEAE-cellulose. F-1 (27 mg),
obtained from gel filtration was applied to the
column (2.1 Â 24 cm) which was pre-equilibrated
with 10 m
M Tris/HCl buffer, pH 8.4 at 4 8C and
eluted by stepwise increases of NaCl concentration
in the same buffer. Insets, polyacrylamide disc gel
electrophoresis of different fractions at room
temperature on 7.5% gel (staining reagent: 1%
amido black).
q FEBS 2001 Lectins from mulberry seeds (Morus sp.) (Eur. J. Biochem. 268) 6007
F-1b (MSL-2) and F-1c (MSL-3), respectively. It was found
that in the presence of 0.1% SDS, MSL-1 gave a single
band, while MSL-2 and MSL-3 gave four distinct bands on
SDS/PAGE (Fig. 4). In the presence of 0.1% SDS and 0.1%
2-mercaptoethanol, MSL-1 gave a strong band correspond-
ing to a molecular mass of 110 000 Da and a weak band
corresponding to molecular mass of 70 000 Da, while the
MSL-2 and MSL-3 gave four bands with molecular masses
of 42 000, 35 000, 25 000 and 19 000, and 35 000, 22 500,
17 000 and 14 950 Da, respectively.
Affinity for Sepharose 4B

the proteins under vacuum. Similar results were obtained
when the concentration of the proteins were measured by the
Lowry method.
The neutral sugar contents of the lectins, MSL-1, MSL-2,
and MSL-3 were found to be 5.7, 5.4 and 4.5%, respectively.
The sugar composition of the lectins as identified by TLC
was found to be glucose and mannose for MSL-1 and
galactose for both MSL-2 and MSL-3.
Cytotoxic effects
All three lectins were found to be toxic and the mortality
rate of brine shrimp nauplii were found to be increased with
concentration of the lectins. As shown in the Fig. 5, the LC
50
(concentration at which 50% mortality of the napulii occurs)
as estimated by extrapolation was 10.96 mg
:
mL
21
for
MSL-1, 17.37 mg
:
mL
21
for MSL-2, and 6.30 mg
:
mL
21
for
MSL-3.
Fig. 3. Ion-exchange chromatography of fraction F-1a on CM-

100% (NH
4
)
2
SO
4
220 3344 15.20 79.06 2.16
Saturated
After gel filtration 80 2842 35.53 67.06 5.04
DEAE-cellulose fraction
F- 1a 12.2 650 53.29 15.37 12.48
F-1b 7.6 818 107.72 19.35 7.56
F-1c 4.8 362 75.38 8.55 15.28
CM-cellulose
F-1a
0
7.3 640 87.98 15.13 10.69
6008 T. Yeasmin et al. (Eur. J. Biochem. 268) q FEBS 2001
Table 2. Hemagglutination inhibition assay of mulberry seed lectins. NI, No inhibition; I, Inhibition.
Proteins Sugar
Concentration (m
M)
InhibitionMaximum Minimum
MSL-1
D-Glucose 110 – NI
D-Mannose 110 – NI
D-Galactose – 20 I
N-Acetyl
D-glucosamine 110 – NI
Methyl-a-

MSL-3 N-Acetyl-galactosamine 110 – NI
D-glucosamine-HCl 110 – NI
Lactose – 20 I
D-Raffinose – 35 I
Fig. 4. SDS/PAGE patterns of the proteins on 10% gel at room
temperature (staining reagent: Coomassie Brilliant Blue). A, F-1a
0
(MSL-) in the presence of SDS; B, F-1a
0
(MSL-) in the presence of SDS
and 2-mercaptoethanol; C, F-1b (MSL-2) in presence of SDS; D, F-1c
(MSL-3) in the presence of SDS.
Fig. 5. Determination of LC
50
of mulberry seed lectins. (W) for
MSL-1 (*) for MSL-2 and (K) for MSL-3.
q FEBS 2001 Lectins from mulberry seeds (Morus sp.) (Eur. J. Biochem. 268) 6009
DISCUSSION
Three lectins have been isolated and purified from a crude
extract of mulberry seeds; the lectins are glycoproteins as
they gave an orange-yellow color in the presence of phenol/
sulfuric acid. The presence of sugar in the lectins was further
confirmed by the findings that they produced a pinkish-red
band on a polyacrylamide gel when the gels are stained with
periodic acid Schiff’s staining reagent after electrophoresis
(data not shown).
The agglutination of rat red blood cells by the lectins
was inhibited specifically in the presence of galactose,
methyl-a-
D-galactopyranoside, methyl-b-D-galactopyrano-

contained mostly four subunits of two identical pairs, e.g.
Indian bean (Dolichos lablab L.) [19], Arbus precatorius
[20], and Ricinus comminis agglutinin [21] and very few
contained four subunits of identical molecular mass, e.g.
Phaseolus vulgaris [3].
The mulberry seed lectins, like those from Abrus
precatorus, Ricinus communis and mistletoe, are toxic in
nature. However it has yet to be determined whether all the
subunits of mulberry seed lectins possess toxicity in addition
to their hemagglutinating properties.
In conclusion, the purified lectins, MSL-1, MSL-2 and
MSL-3, besides being specific for rat red blood cell
agglutination, are members of the galactose-binding lectins.
ACKNOWLEDGEMENTS
The authors thank Dr Abdul Aziz Sarkar, Senior Scientific Officer,
Bangladesh Sericulture Research and Training Institute, Rajshahi,
Bangladesh for supplying mulberry seeds during the period of the
research.
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