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ISOLATION AND STUDY OF PHYSICOCHEMICAL
PROPERTIES OF LECTIN EXTRACTED FROM
PHASEOLUS
VULGARIS
SEEDS
Rashidova Nodira Qobiljon qizi
Tashmukhamedova Shoxista Sabirovna
Tashkent Pharmaceutical Institute
*e-mail: rashidovan93@gmail.com
https://doi.org/10.5281/zenodo.16908498
ARTICLE INFO
ABSTRACT
Received: 13
th
August 2025
Accepted: 18
th
August 2025
Online: 19
th
August 2025
This study investigates the extraction and characterization of
lectin from Phaseolus vulgaris seeds. Lectins are
carbohydrate-binding glycoproteins known for their wide
range of biological activities, including antimicrobial and
anticancer effects. The seeds were extracted in phosphate-
buffered saline, and incubation time was optimized.
Maximum protein yield (42.7 mg) was obtained after 10
hours. Ammonium sulfate at 75–80% concentration was most
effective for protein precipitation, coinciding with peak
hemagglutination activity. Purification was performed using
gel filtration on a Sephadex G-75 column. The biological
activity of lectin was tested at different temperatures and pH
levels. High activity was observed at 20–25°C and pH 5.0–8.0.
SDS-PAGE analysis revealed the molecular weight of lectin to
be around 62–63 kDa. The findings confirm that Phaseolus
vulgaris lectin is a stable and active protein with promising
applications in biotechnology and pharmaceutical research..
KEYWORDS
Phaseolus vulgaris, lectin,
bioactivity.
Relevance of the Topic.
Lectin is a glycoprotein that binds specifically to carbohydrates
such as mannose, galactose, fucose, and rhamnose. Depending on the type of carbohydrate base,
lectins can exhibit a wide range of biological activities. To date, lectins have been widely used
in medicine, diagnostics, pharmaceuticals, and agriculture.
Plant-derived lectins have shown antibacterial, antifungal, antiviral (including against the
human immunodeficiency virus), anticancer, anti-inflammatory, and analgesic properties.
These lectins can be extracted from various parts of plants, including leaves, seeds, stems, roots,
and fruits. Among plants, legumes are considered one of the main sources for lectin extraction.
However, it has been found that many lectins derived from legumes can irritate the
intestinal villi in humans, making them unsuitable for direct consumption in food. Therefore,
thorough investigation is required before they can be used safely in medicine. [1-4]
Materials and Methods.
Dried
Phaseolus vulgaris
seeds were ground into powder and
passed through a 280-micron sieve. The resulting powder was mixed with phosphate-buffered
saline (PBS, 10 mM, pH 7.0) and placed on a magnetic stirrer overnight at 4°C. The extract was
then filtered and centrifuged. Ammonium sulfate was gradually added to the supernatant until
saturation, and the solution was dialyzed in a dialysis bag for 24 hours to achieve desalting.
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The obtained lectin was further purified using gel filtration. A 10 mg/mL solution of
deionized lectin was filtered through a 0.45 µm membrane and subjected to gel filtration on a
Sephadex G-75 column using Tris-HCl buffer at a flow rate of 1.5 mL/min. The lectin-containing
fractions were lyophilized to obtain a dry powder. [5-6]
Determination of Hemagglutination Activity.
Hemagglutination activity was tested
using specialized Szyan titration microplates. A 0.5 mg/mL solution of lyophilized lectin was
prepared. 50 µL of this solution was serially diluted with physiological saline, and 5 µL of a 2%
rabbit erythrocyte suspension was added. The reaction was incubated for 45 minutes at room
temperature.[3]
Results and Discussion.
The optimal conditions for lectin extraction were identified. The
table below summarizes the impact of incubation time on protein yield:
1-table
The effect of incubation time on the lectin extraction process
№
Volume of extract
(ml)
Incubation Time
(hours)
Volume of protein
(mg)
1
750
2.0
29.1
2
750
4.0
36.1
3
750
6.0
38.6
4
750
8.0
41.4
5
750
10
42.7
6
750
12
42.7
These results indicate that increasing the incubation time has a positive effect on protein
extraction, with the maximum protein concentration (42.7 mg) observed after 10 hours, which
remained stable afterward.
In the next stage, vacuum filtration (Nutch method) was used to process the extract. After
centrifugation (7000 rpm, 20 minutes), the supernatant was subjected to ammonium sulfate
precipitation. The hemagglutination activity was highest when using 75–80% ammonium
sulfate, which was identified as the optimal concentration for protein precipitation.
Additional Biological Activity Tests.
Agglutination tests with trypsin-treated human
erythrocytes confirmed strong lectin activity. Microscopy images showed erythrocyte clumping
after 15 minutes, indicating strong hemagglutination. Thermal stability tests revealed high
activity between 10°C–25°C; activity decreased at 50–60°C. pH stability tests showed maximum
activity at pH 5.0–8.0.
The effect of incubation time on the extraction process was studied.
As shown in the table, the extraction process is dependent on time. An increase in incubation
time positively influenced the efficiency of lectin extraction. The data demonstrate that after 10
hours of incubation, the protein content reached
42.7 mg
, and this amount remained
unchanged with further incubation.
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In the next stage of the study, the extract was filtered using the
Nutsch vacuum filtration
method.
The extract was poured gradually and filtered in 50 mL portions, with the filter paper
replaced after each portion. The process continued until all extract was processed.
The resulting filtrate was centrifuged at
7000 rpm for 20 minutes
, and the supernatant
was separated. Proteins from the supernatant were then precipitated using
ammonium
sulfate.
The
optimal conditions for precipitation
were also determined in the course of this process.
1- graph
Effect of ammonium sulfate concentration on the precipitation of lectin from the
extract
As shown in the linear graph above, the hemagglutination activity of lectin was used to
determine the optimal concentration of ammonium sulfate for protein precipitation from the
extract. The highest protein precipitation and a simultaneous increase in hemagglutination
activity were observed at ammonium sulfate concentrations between 75% and 80%. Based on
these results, this concentration range was identified as optimal for the precipitation of lectin
from the
Phaseolus vulgaris
extract.
Determination of Lectin Activity via Hemagglutination.
The biological activity of the
lectin was assessed through agglutination testing. Trypsin-treated human erythrocytes were
used in specialized microplates for the hemagglutination assay.
To perform the test, 50 µL of phosphate-buffered saline (PBS) was added to each well,
followed by serial two-fold dilutions of the lectin extract (10 dilution steps). Next, 50 µL of a 2%
erythrocyte suspension was added to each well. The mixtures were incubated at room
temperature for 1 minute to 20–30 minutes and observed under a microscope.
The extent of agglutination was determined by visually assessing the clustering of red
blood cells. If no agglutination occurred, the erythrocytes remained dispersed individually.
If agglutination was present, the red blood cells appeared clumped together, indicating
lectin binding activity.
0
10
20
30
40
50
60
70
80
90
5
Hema
gg
lu
tina
tio
n
acti
vit
y%
Ammonium sulfate concentration, (%)
10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
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Figure 1. Microscopic appearance of the hemagglutination process
A – Initial state of erythrocytes before agglutination.
B – State after 15 minutes of incubation showing visible cell agglutination.
Study of the Effect of Temperature on Lectin Activity.
The graph below illustrates the
dynamics of changes in the hemagglutination activity of lectin at different temperatures over
various time intervals. This allows for the assessment of how temperature influences the
biological activity of lectin.
2-graph
Effect of temperature on lectin activity
This graph demonstrates that lectin activity is temperature-dependent. Hemagglutination
activity was measured at various temperatures — 10°C, 20°C, 30°C, 40°C, 50°C, and 60°C — and
across different time intervals (10, 20, 30, and 40 minutes) for each sample.
The results indicate that lectin retains high biological activity between 10°C and 20°C
throughout the 10–40minute incubation period. However, as the temperature increases, a
negative correlation is observed between temperature and lectin activity. Both prolonged
incubation time and higher temperatures lead to a gradual decrease in hemagglutination
activity.
0
20
40
60
80
100
120
10
20
30
40
50
60
He
ma
ggl
utin
ation
a
ctivity%
Temperature, C
10'
20'
30'
40'
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A sharp decline in activity is particularly noticeable at 50°C and 60°C, even within short
exposure times. These findings confirm that lectin maintains its stability and biological activity
up to approximately 25°C.
Study of the Effect of pH on Lectin Activity
The hemagglutination assay of lectin under various pH conditions demonstrates that the
lectin remains active within a pH range of 3.0 to 11.0, as shown in graph 3.
3
-
graph
Study of the Effect of pH on Lectin Activity
Lectin hemagglutination activity increased with rising pH levels and reached its maximum
between pH 5.0 and 8.0. However, in alkaline conditions ranging from pH 10 to 11, the activity
dropped significantly, maintaining less than 50% of its initial agglutination capacity. These
results indicate that both extremely acidic and highly alkaline environments adversely affect
the hemagglutination activity of lectin.
Purification of the Isolated Lectin Compound.
It is well known that gel filtration
chromatography can be used to remove unwanted low-molecular-weight impurities from
protein preparations. In this study, gel filtration was employed to purify the isolated lectin by
eliminating foreign iodinated substances. The purification process was carried out using the
Bio-Rad BioLogic LP chromatography system.
The column was packed with Sephadex G-75 gel, and the flow rate of the Tris-HCl buffer
was set at 1.5mL/min. The results of the chromatographic purification are shown in the figure
below.
0
20
40
60
80
100
120
1
2
3
4
5
6
7
8
9
10
11
12
13
14
He
maggl
utin
ati
on
a
cti
vity%
pH
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Figure 2. Chromatogram of lectin isolated from
Phaseolus vulgaris
, obtained using
the Bio-Rad BioLogic LP chromatography system
The marked area in this figure represents the fraction that exhibited high
hemagglutination activity.
SDS-PAGE Analysis of Lectin. The electrophoretic analysis of dry lectin samples on
polyacrylamide gel was carried out using the Laemmli method [1]. The procedure was
performed using the Mini-PROTEAN II Electrophoretic Cell (BIO-RAD, USA).
Two types of gels were prepared:
Resolving gel: Tris-HCl buffer (pH 8.8) 244 mM; Bis-acrylamide 10%; TEMED 0.08%; APS
0.08%; SDS 0.01%
Stacking gel: Tris-HCl buffer (pH 6.8) 125 mM; Bis-acrylamide 4%; TEMED 0.05%; APS
0.05%; SDS 0.01%
The electrode buffer consisted of Tris-OH 25 mM, glycine 192 mM, and 0.2% SDS.
Sample preparation included 60 μg of protein in 20 μL volume. Before loading onto the
gel, samples were denatured at 90°C for 3 minutes in a solution containing 0.004%
bromophenol blue (Sigma, USA), 10% mercaptoethanol, and 4% SDS.
After electrophoresis, the gels were stained with Coomassie Brilliant Blue G-250 aqueous
solution. The destaining buffer consisted of acetic acid, ethanol, and water in a 1:1:8 ratio.
The electrophoresis was run under the following current conditions:
40 mA during stacking phase
80 mA during resolving phase
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Figure 3. Molecular weight of lectin isolated from
Phaseolus vulgaris
determined by
SDS-PAGE
As shown in Figure 3, the molecular weight of the isolated lectin was found to be
approximately 62–63 kDa.
Conclusion.
In this study, a series of experimental steps were conducted to extract, purify,
and characterize the physicochemical properties of lectin derived from the seeds of
Phaseolus
vulgaris
(common bean). Based on the results obtained, the following conclusions were drawn:
The optimal incubation time for protein extraction was determined to be 10 hours, during
which the maximum protein content in the extract reached 42.7 mg;
The most effective ammonium sulfate concentration for protein precipitation was found
to be 75–80%, corresponding to the highest hemagglutination activity;
The biological activity of lectin was shown to be dependent on temperature and pH.
Maximum activity was observed at 20–25°C and in the pH range of 5.0 to 8.0. Elevated
temperatures and extreme acidic or alkaline conditions significantly reduced activity;
The purified lectin was successfully separated using Sephadex G-75 gel filtration, and
fractions with high hemagglutination activity were isolated;
SDS-PAGE analysis revealed that the molecular weight of the lectin is approximately 62–
63 kDa.
These findings suggest that the lectin extracted from
Phaseolus vulgaris
seeds possesses
high biological activity and may serve as a promising candidate for further exploration in the
fields of pharmaceuticals and biotechnology.
References:
1.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of
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EURASIAN JOURNAL OF MEDICAL AND
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2.
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