Identification of Hemoglobin Variants in Unknown Mixtures

This experiment aims to identify the type of hemoglobin present in unknown mixtures of two samples. The experiment involves the determination of three standards containing hemoglobin variants and two unknown mixtures of hemoglobin samples. The document discusses the results and implications of the experiment.

Running head: HUMAN GENETICS
Topic: HUMAN GENETICS
Name of the Student:
Name of the University:
Author Note:

1HUMAN GENETICS
Aim:
The aim of the experiment is the determination of the type of hemoglobin which is
present in the unknown mixture of two samples (Hb mix1 and Hb mix2). Three standards
would be provided containing hemoglobin variants and two unknown mixtures of
hemoglobin samples have been provided which has to be identified. The three variants which
has been provided include HbA, HbO and HbA2. These are the non-glycosylated native
proteins which are mainly present in the human body and have been compared with HbS
strains which are the hemoglobin present in patients suffering from sickle cell anemia.
Introduction:
According to Srivastava et al (2014), hemoglobin is defined as a protein rich in iron
and present in blood cells. Hemoglobin is the pivotal protein for carrying oxygen to the
tissues within the body and the main reason for attaining the disc shape of the red blood cells.
Cyanmethamoglobin methods are usually applied for the quantification of hemoglobin.
Sickle cell anemia has been a disorder due to a mutation in the amino acid sequence.
According to Parrow et.al (2017), decrease in erythrocyte deformability is one of the reasons
for severity of the disease in Sickle Cell Anemia (SCA). The heterogeneity of red cells along
with deformability is also an influencing factor for the concentration of alternate hemoglobin
like fetal hemoglobin (HbF) as well as the adult variants of HbA2 and HbAO. Processes like
deformability as well as osmolality including index minimum for elongation near hypotonic
region is one of the important factors for monitoring fetal hemoglobin in people suffering
from sickle cell anemia. HbA2 is the normal variant of hemoglobin consisting of the alpha
and beta chains where anomalies may occur in thalassemia due to structural changes (Ivaldi
et al. 2014).

2HUMAN GENETICS
HbC is the kind of cells found in the patients who suffer from sickle cell anemia with
homozygous CC alleles. CC diseases are mainly characterized by increased mean corpuscular
concentration of hemoglobin. It is different from the SS variety or HbS where dense cells are
important in representation of a specific fraction of the cells. Among the comparison of HbA,
HbS and HbC binding to the red membrane cells and the subsequent amount of hemoglobin
present in the mixture of hemoglobin variants is the order of HbC >HbS>HbA (Gonclave,
Gupta and Penman 2016). According to Booth et al (2015), HbA is the most common form of
adult tetramer constituting almost 97% of the levels of hemoglobin found in the body. Like
HbA2 it consists of two alpha and beta chains.
Hemoglobin electrophoresis is often used as a screening test for the identification off
abnormal and variant genes which mainly includes Hemoglobin A1, hemoglobin A2
hemoglobin F and hemoglobin C as well as Hemoglobin S. Total hemoglobin is a mixture of
the various components of the Hb and has a negative charge. With application of electric
potential they migrate to the cathode and anode depending on the charge. Usually, in gel
electrophoresis done with the hemoglobin sample migration occurs in the order HbA2, HbC,
HbS, HbF, HbA. Thus from the migration the usual distance of migration involving HbF and
HbC is estimated. Various factors affecting the migration of these bands include correct pH,
proper concentration of the buffer used and the temperature of the buffer used in the
experiment (Piety et al. 2015).
HbF or fetal hemoglobin is the most prominent form of hemoglobin which is
expressed in the developing fetus. There is a difference of the alpha and beta chains
constitution in adult and fetal hemoglobin which contribute mainly to the structural
differences. The primary differences are located near the 2,3 BPG binding sites which are
present between the gamma 1 and gamma 2 interface of HbF and the beta 1 interface of HbA
(Donelly et al. 2014).

3HUMAN GENETICS
There is a distinct difference in terms of structure between normal and affected
individuals. Apart from conformational changes, there is a difference in the structure and PI
(isoelectric point) which is responsible for the differences in various attractive interactions
which is responsible for the filamentation of HbS leading to its structural anomalies.
Differences can be seen from spectrophotometry studies which results in the self-assembly of
the HbS fibrils producing sickling of red blood cells (Openi.nlm.nih.gov, 2019). Sickle cell
anemia occurs due to the A-T which is single base substitution and usually occurs in the first
exon of the beta globin gene. There is a relation of the disease with genetic mutation as
pathophysiological consequences mainly originate from the monogenic defect. Moreover, the
substitution of amino acids in Beta globin which allows the formation of tetramers of
defective hemoglobin which polymerize upon deoxygenating. Despite being a monogenic
diseases sickle cell anemia is variable in its clinical severity (Hoban, Orkin and Bauer 2016).
Sickle cell anemia has a profound effect on red blood cells which form a sickle like shape due
to the mutation in Glu7Val which ultimately changes the shape into a rigid curved structure
which blocks the passage of he vasculature. The change in structure become susceptible to
hemolysis causing an increase in the plasma levels of free Hb (Eshbach et al. 2017).
The most important hematological finding is the increased levels of HbA2. Thus the carriers
of Beta thalassemia have unusual increased levels of HbA2. Point mutations are very
common in these carriers often involving the 5’ promoter region of the beta gene (Galanello
2015).
Results
Electrophoresis of hemoglobin variants help in the segregation of the various varieties of the
hemoglobin including the normal and affected strains which are based on the positive or
negative charges of the components and their relative migrations based on the charges. The

4HUMAN GENETICS
relative migrations of the variants are given which is 9 mm for HbAO, 4 mm for HbA2, 6
mm for HbS. From the migration distances of the unknown mixture it can be seen that the
migration distances are 4 mm and 9mm which is he distance for HbAO and HbA2. Similarly
for the unknown mixture the migration distances are 6 m and 9 mm. These again are similar
to the distances shown in HbS and HbAO .Thus from the unknown mixture of various
hemoglobin variant samples the components have been separated on the basis of their
respective charges and thus, it can be interpreted that the first mixture contains HbA2 and
HbAO whereas the second sample contains HbS as well as HbAO. The difference in
migration due to the different charge depends on the composition of various amino acids.
Additional positive charges are contributed through nitrogenous groups while negative
charges are due to the contribution of second carboxyl group of aspartic and glutamic acids
(Khosa et al. 2015). From the migration order (HbA2> HbC> HbS> HbF> HbA) it can be
seen that the possible migration of HbC will be between 6 mm and 4 mm and the difference
between the distance for HbF will be between 9m and 6mm. Hence, the net positive charge
on sickle cell anemia is due to the presence of large number of positively charged nitrogenous
groups with the normal. These kind of tests are used for patients who need blood transfusion
for the detection of impure blood.
Discussion
Sickle cell anemia has definite effects on the body. The clinical manifestations of sickle cell
anemia affect almost all organs and systems around the body. Systemic inflammatory state as
well as chronic hemolytic anemia for the basic pathophysiological mechanism for vaso-
oclusive vents .The effects of sickle cell anemia on an individual may be short term as well as
long term like end organ damages of chronic kidney disease heart damage retinopathy
pulmonary hypertension and osteoporosis (Costa and Frankin 2016).

5HUMAN GENETICS
Sickle cell anemia has a profound effect on the lifelong inherited conditions affecting the
hemoglobin. Considering populations around the world, most people affect are of the African
or Caribbean origin and according to the statistical estimates, there are almost 12,500 and
15,000 people who have the sickle cell diseases in UK. Management for such diseases is
variable all around UK and often a frequent reason for complaints (Nice.org.uk. 2019).

6HUMAN GENETICS
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