Hemagglutinating specificity of Lection isotopes from

Phaseolus vulgaris (String beans) on

different blood groups

Submitted by;
Digman, Alexix B.
Ruiz, John Michael F.
Vicedo, Ma. Zhairene R.

Submitted to:
Ms. Laarni E. Gloriani

CHAPTER I
INTRODUCTION
Beneficial and edible substances are available in nature in terms of food industry.
It only does not give nutritious effects to an individual, but also it can be used in
research purposes for it contains special molecules that can be helpful in medical
field.

Phaseolus vulgaris, also known as Sitaw in the Philippines is an important bean

worldwide. It is commonly present in Philippines, and other top bean grower such as
Asia, Africa, Europe, Oceania, South and North America. P. vulgaris can be an
excellent source of micronutrients like iron, zinc, thiamin and folic acid (Petry et.al,
2015) and it is also high in potassium, selenium, molybdenum, and vitamin B6.

While public knows its benefits only in nutrients it provides to human, other
researches found another way how this particular bean can contribute not only in food
industry but also in medical field. P. vulgaris contains lectin, which can also be
extracted from other sources like fruits, vegetables, nuts and grains.

Lectin is a glycoprotein famous for its sugar-binding capacity. It was studied and
used as molecular tools for the study of carbohydrate architecture and dynamics on
the cell surface (Jelil & Jabor, 2012). A specific amount of this can be an excellent
reagent for anti-A, anti-B, and anti-N (Khan, 2006). It contains specialized functions
which can agglutinate red blood cells and forms antigen-antibody complex when
exposed to its particular sites, called hemeagglutination (Jebor & Jalil, 2012). Thus
the researcher thought of a study which will determine the hemagglutinating
specificity of lection isotopes from Phaseolus vulgaris (String beans) on different
blood groups.

A. Statement of the Problem

1. What is the level of agglutination of lectin isotopes from Phaseolus vulgaris

on different blood groups?
2. What blood groups are agglutinated by Phaseolus vulgaris (String beans)
extract?
3. Is there a significant difference with the agglutination capacity using
Phaseolus vulgaris (String beans) and prepared blood typing sera?

B. Significance of the Study:

For future Researchers:
1. To compare antigenic specificity of the purified Lectin isotopes towards
various blood groups.
2. To establish general knowledge about Lectin, its isotopes, and the
corresponding

blood

groups

they

agglutinate

in

the

field

of

Immunohematology for future studies.

For Blood Bank personnel:

1. To secure other sources of Lectin for hemagglutinating studies on Blood
Banking.

CHAPTER II
REVIEW OF RELATED LITERATURE

Lectins
Lectins are defined as proteins or glycoproteins possessing at least one noncatalytic domain which binds reversibly to a specific mono- or oligosaccharide (Jebor
and Jalil 2012). The word Lectin has been derived from the Latin word which means
I choose, because lectins are very specific to the site to which it binds (Bhol 2012).
Lectins are widely distributed in nature and found in all forms of life including plant
products such as fruits, vegetables but nuts, grains, beans and seeds contains high
lectin amount (Lis and Sharon 1986). Researchers have great interest and lectins have
been studied and isolated from various sources including plants , animals, fungi,
lichens and bacteria (Liener 1976).

Because of their sugar binding properties, lectins have been extensively studied
and used as molecular tools for the study of carbohydrate architecture and dynamics
on the cell surface (Jebor and Jalil 2012). This specific trademark of lectins grants
itself as carbohydrate-binding proteins which bind to glycoproteins, glycolipids, and
also polysaccharides (Goldstein and Hayes 1978). These proteins can be classified
into many groups such as mannose-binding, glucose-binding, galactose-binding, Nacetylglucosamine- binding, N-acetylgalactosamine- binding, fucose-binding, and
sialicacid-binding (Bhol 2012). In addition, they have non-immune origin and can
bind to the above mentioned biochemical compounds without changing their covalent
structure (Kocourek & Horejsi 1981; Barondes 1988).

Lectins can be used as probes for the characterization and isolation of simple and
complex sugars (Rudiger and Gabius 2001). Furthermore, these proteins were
characterized for their agglutination properties with erythrocytes of human and other
animals, it is the easiest and most convenient method of detection of lectin activity
(Laija et al. 2010). As antigenic determinants of blood group, these proteins have
come to be important tools in the identification of different blood groups antigens. A

handful of lectins may be considered excellent reagents for anti-A, anti-B, anti-N etc,
(Khan 2006). Isotopes of these proteins along within antibodies may be used as
markers for the detection of basal cells in the human respiratory epithelium.
Furthermore, they suggest that the glycosylation of some glycocomponents of the
basal cells is under the control of the genes of the secretor- and ABO-blood group
system (Bals and Welsch 2003).

One of the major sources of plant lectins, family fabaceae or leguminosae is a

plant group that is characterized by its stipulated leaves and its fruit (beans). This
major family under kingdom plantae comes from the latin word faba which means
beans (Lavin and Sanderson, 2014). The ovary of this plant family develops into a
legume - a simple dry fruit that usually opens along a seam on two sides commonly
named as "pod". The family Fabaceae has an essentially worldwide distribution, being
found everywhere except Antarctica and the high arctic regions. The trees are often
found in tropical regions, while the herbaceous plants and shrubs are predominant
outside the tropics (Schrire, Lewis, Lavin 2005). Over 730 known genera are present
around the world, among which are few established studies on the use of lectin in the
field of immunohematology.

Phaseolus vulgaris
Among the known genera of lectin-containing legumes is Phaseolus vulgaris. P.
vulgaris (also known as Sitaw, common bean, string bean, field bean, flageolet bean,
French bean, garden bean, green bean, haricot bean, pop bean, or snap bean) is a
herbaceous member of the family Fabaceae known for its edible dry seed. This plant
species is of worldwide distribution and is biogeographically abundant in tropical
regions such as the Philippines.
The conjugation of particulate test antigens to a carrier which could be artificial
(latex or charcoal particles) or biological (red blood cells) is responsible for
agglutination reactions. Patient serum seemingly containing antibodies are reacted to

these conjugated particles which has an endpoint of observation of clumps due to the
formation of antigen-antibody complex. The time of incubation with the antibody
source, amount and avidity of the antigen conjugated to the carrier, and conditions of
the test environment (e.g., pH and protein concentration) determines the quality of the
test result. In diagnostic immunology, various methods are used such as latex
agglutination, flocculation tests, direct bacterial agglutination, and hemagglutination
(Boundless, 26 May 2016).

The isolation of lectin is essential in this experiment for without this, its
physiological activity present in the natural sources will not be fully appreciated. In
order to properly express the hemagglutinating property (Jebor & Jalil, 2012) of lectin
in vitro, it must be first isolated from its sources. There are plenty of methods on how
to extract lectin from beans some are the traditional ones.

Affinity chromatography by a Sepharose-4B column was used to purify the

lectin from the legume of choice. The goal is to know the elution profile of the
legume lectin from lactamyl-sepharose affinity matrix for 60% and 90% affinity
(Chandra, 2012). The stationary phase is typically a gel matrix (sepharose) which is a
sugar molecule derived from an algae. The starting point I purification is an undefined
heterogeneous group of molecules in solution, such as a plant cell lysate in this case.
A well-known and defined property is to be exploited during the affinity purification
process for the molecule of interest. It is an entrapment process wherein the target
molecule is being trapped on a solid or stationary phase or medium. The specific
property will separate the lectin from the undesired molecules in the mobile phase.
The unwanted molecules will not become trapped as they do not possess this property.
The stationary phase can then be removed from the mixture, washed and the target
molecule released from the entrapment in a process known as elution (Uhlen, 2008).
Affinity chromatography is performed for the purification of recombinant proteins
and biochemically diverse molecules such as Lectins. Overall, the uses of affinity

chromatography are: to purify and concentrate a substance from a mixture into a

buffering solution; reduce the amount of a substance in a mixture; discern what
biological compounds bind to a particular substance; and purify and concentrate an
enzyme solution properties that are ideal for purifying a complex biochemical
compound such as Lectin by elimination of unwanted molecules.

CHAPTER III
METHODOLOGY
This chapter shall discuss the methodology available in the study and what is
applicable to use for the proper execution of answers in the statement of problems in
chapter I which is to determine the hemagglutinating activity of lectin isolated and

purified from Phaseolus vulgaris on different blood groups.

Likewise, this chapter talks about the various methods on how to perform the
complex processes included on the study and how to achieve accurate data.

For this study, the experimental research method was utilized. In this method, all
variables are manipulated to get the desired results including the numbers of string
beans used and numbers of random blood specimen used.

SAMPLE COLLECTION:
The String beans (Phaseolus vulgaris) can be obtained anywhere in the
Philippines. It is widely available in the market for consumption use.

ISOLATION OF PHASEOLUS VULGARIS LECTIN:

I. Traditional Methods

A. Soaking Method
String beans were ground to a powder in an electric mill and filtered through 80
mesh grit. The powder (5g) was mixed with 0.15M NaCl (1:8, w/v) for 48 h at 4C,
and filtered through 80 mesh grid. Subsequently, the filtrate was centrifuged at 9168
xg for 30 minutes, and the supernatant was fractionally precipitated with ammonium
sulfate at 40%, 50%, 60%, and 70% saturation, respectively. The four pellets were
combined, dissolved in a minimal volume of water, and dialyzed against distilled
water at 4C.

B. Degreasing Method
The powder was defatted twice with Sherwood oil (1: 8, w/v) for 4 h at 4C,
which was removed by volatilization at room temperature, to obtain degreased bean
powder. The degreased bean powder was mixed with 0.15M NaCl (1:8, w/v) for 48 h

at 4C, and subsequent procedures were identical to those described in the soaking
methid.

(3) Homogenate Method

Red kidney beans (5g) were softened in 400mL NaCl (1:8, w/v) for 24 hours at
4C, then homogenized in a blender. The homogenate was diffused for a further 24
hours at C, then filtered through an 80 mesh grid. The filtrate was centrifuged at 9168
x g for 30 minutes, and the supernatant was precipitated hierarchically by ammonium
sulfate.

II. Reversed Micelle Extraction

The reverse micellar system comprised sodium di-(2-ethylhexyl) sulfosuccinate

(AOT) in isooctane. Extraction and back extraction assays were performed by phase
contact 1:1 (v/v), stirred for 5 minutes and separated by centrifugation at 2292 xg for
5 minutes (Nascimento et al.).

III. Determination of Protein Concentration

Bradfords method was used for protein quantification, using bovine serum
albumin (BSA) as the standard. The relative protein concentration of the eluted
fractions was determined by measuring the absorbance at 280nm.

sIV. Hemagglutinating Activity Assay

Serial two-fold dilutions of the lectin solution in microtiter v-plates (25 L) was
mixed with 25L 2% chicken red blood cell suspension in saline (pH 7.2). Readings
were recorded after about 30 minutes at room temperature, when the blank had fully
sedimented. The hemagglutination titer, defined as the reciprocal of the highest
dilution exhibiting hemagglutination, was treated as one hemagglutination unit.

Specific activity was expressed as the number of hemagglutination units per mg

protein.

V. Purification of String Bean Lectin

Crude extract from reversed micelles was dialyzed against 0.1M NaCl in 0.02M
Tris-HCl (pH 8.0), loaded on a DEAE Sephadex A-50 column (2.6 cm 60 cm) that
had been equilibrated with the same buffer, and subjected to ion exchange
chromatography. The column was washed initially with 0.1M NaCl in 0.02 M TrisHCl (pH 8.0) to remove proteins that had not specifically absorbed to the column,
then washed with linear salt gradient elution. Fractions showing hemagglutinating
activity were further purified by sieve chromatography on a Sephadex G150 column
in 0.15M NaCl in 0.02 M Tris-HCl (pH 8.0).

VI. SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was

performed (Laemmli et al.), using a 15% separating and a 5% stacking gel.