Hemoglobin Electrophoresis (Biochemistry)

Overview of Hemoglobin Overview of Hemoglobin Electrophoresis Electrophoresis

Normal Hemoglobin Normal Hemoglobin StructureStructure

Hemoglobin A is a tetramer Hemoglobin A is a tetramer composed of 4 subunits:composed of 4 subunits:– 22αα and 2 and 2ββ

Each subunit has a ring (porphyrin Each subunit has a ring (porphyrin ring) which holds an iron molecule. ring) which holds an iron molecule. – This is the binding site of oxygenThis is the binding site of oxygen


Hemoglobin tetramer


Fe

O

O

The oxygen atom binds to the Fe atomperpendicular to the porphyrin ring

Porphyrin ring O2 binding site

Hemoglobin FunctionHemoglobin Function

The function of the Hemoglobin The function of the Hemoglobin molecule is to pick up oxygen in the molecule is to pick up oxygen in the lung and deliver it to the tissues lung and deliver it to the tissues utilizing none of the oxygen along utilizing none of the oxygen along the way. the way.

Hemoglobin FunctionHemoglobin Function

The normal hemoglobin molecule is well The normal hemoglobin molecule is well suited for its functionsuited for its function– Allows for OAllows for O2 2 to be picked up at high Oto be picked up at high O22 tension tension

in the lung and delivered to the tissues at low in the lung and delivered to the tissues at low OO2 2 tension.tension.

– The oxygen binding is cooperative:The oxygen binding is cooperative: As each OAs each O22 binds to hemoglobin, the molecule binds to hemoglobin, the molecule

undergoes a conformational change increasing the undergoes a conformational change increasing the OO22 affinity for the remaining subunits. affinity for the remaining subunits.

This creates the sigmoidal oxygen dissociation curveThis creates the sigmoidal oxygen dissociation curve

Normal Hemoglobin Normal Hemoglobin FunctionFunction

The hemoglobin dissociation curveThe hemoglobin dissociation curve

Normal Hemoglobin Normal Hemoglobin FunctionFunction Many variables influence the dissociation curve:Many variables influence the dissociation curve:

– pH:pH: An increase in pH (dec. COAn increase in pH (dec. CO22) shifts the curve to the left ) shifts the curve to the left

(increased O(increased O22) affinity) affinity A decrease in pH (inc. COA decrease in pH (inc. CO22) shifts the curve to the right ) shifts the curve to the right

(decreased O(decreased O22 ) affinity ) affinity

– Temperature:Temperature: Increased temp with increased metabolic demands causes Increased temp with increased metabolic demands causes

decreased Odecreased O2 2 affinity (right shift) and increased Oaffinity (right shift) and increased O22 delivery delivery

– 2,3 DPG (2,3-diphosphoglycerate):2,3 DPG (2,3-diphosphoglycerate): Lowers OLowers O2 2 affinity by preferentially binding to affinity by preferentially binding to BetaBeta chain of chain of

deoxyhemoglobin, stabilizing it and reduces the intracellular deoxyhemoglobin, stabilizing it and reduces the intracellular pHpH

– As hemoglobin concentration decreases, 2,3 DPG increases, As hemoglobin concentration decreases, 2,3 DPG increases, allowing more Oallowing more O22 to be unloaded to be unloaded

Hemoglobin variantHemoglobin variant

Hemoglobin variants are mutant forms of Hemoglobin variants are mutant forms of hemoglobin in a population (usually of hemoglobin in a population (usually of humans), caused by variations in humans), caused by variations in genetics.genetics.

Some hemoglobin variants such as Some hemoglobin variants such as sickle-cell anemia causes diseases, sickle-cell anemia causes diseases, hence they are hemoglobinopathies. hence they are hemoglobinopathies. Other variants cause no detectable Other variants cause no detectable disease, thus considered non-disease, thus considered non-pathological variants. pathological variants.

Other Hemoglobins in normal Other Hemoglobins in normal adults (non pathological)adults (non pathological)

HemoglobinHemoglobin StructureStructure %%

AA αα22 ββ22 92%92%

AA22 αα22 δδ22 2.5%2.5%

AA1C1C αα22 ( (ββ-N-glucose)-N-glucose) 3%3%

FF αα22 γγ22 <1%<1%

Gower-1Gower-1 ζζ22 εε22 0*0*

Gower-2Gower-2 αα22 εε22 0*0*

PortlandPortland ζζ22 γγ22 0*0** Indicates early embryonic form not seen in adults

Hemoglobin AbnormalitiesHemoglobin Abnormalities

There are 3 main categories of inherited There are 3 main categories of inherited Hemoglobin abnormalities:Hemoglobin abnormalities:– Structural or qualitative: The amino acid Structural or qualitative: The amino acid

sequence is altered because of incorrect DNA sequence is altered because of incorrect DNA code (Hemoglobinopathy).code (Hemoglobinopathy).

– Quantitative: Production of one or more globin Quantitative: Production of one or more globin chains is reduced or absent (Thalassemia).chains is reduced or absent (Thalassemia).

– Hereditary persistence of Fetal Hemoglobin Hereditary persistence of Fetal Hemoglobin (HPFH): Complete or partial failure of (HPFH): Complete or partial failure of γγ globin globin to switch to to switch to ββ globin. globin.

Hemoglobinopathy?Hemoglobinopathy?

Hemoglobinopathy is a genetic Hemoglobinopathy is a genetic defect that results in abnormal defect that results in abnormal structurestructure of one of the globin chains of one of the globin chains of the hemoglobin molecule.of the hemoglobin molecule.

Causes the Causes the depletion in the synthesis depletion in the synthesis of sufficient normal hemoglobinsof sufficient normal hemoglobins

Pathological variantsPathological variants

1.1. Hemoglobin H (Hemoglobin H (β4) - β4) - A variant A variant form of hemoglobin, formed by a form of hemoglobin, formed by a tetramer of tetramer of β β chains, which may be chains, which may be present in variants of present in variants of α α thalassemia.thalassemia.

2.2. Hemoglobin Barts (Hemoglobin Barts (γ4) - γ4) - A A variant form of hemoglobin, formed variant form of hemoglobin, formed by a tetramer of by a tetramer of γ γ chains, which may chains, which may be present in variants of be present in variants of α α thalassemia.thalassemia.

Pathological variantsPathological variants 3.3. Hemoglobin S (Hemoglobin S (α2βα2βS2) - A variant S2) - A variant

form of hemoglobin found in people with form of hemoglobin found in people with sickle cell disease. There is a variation sickle cell disease. There is a variation in the in the β-β-chain gene, causing a change in chain gene, causing a change in the properties of hemoglobin, which the properties of hemoglobin, which results in sickling of red blood cells.results in sickling of red blood cells.

4.4. Hemoglobin C (Hemoglobin C (α2βα2βC2) - Another C2) - Another variant due to a variation in the variant due to a variation in the β-β-chain chain gene. This variant causes a mild chronic gene. This variant causes a mild chronic hemolytic anemia.hemolytic anemia.

Pathological variantsPathological variants 5.5. Hemoglobin E (Hemoglobin E (α2βα2βE2) - Another E2) - Another

variant due to a variation in the variant due to a variation in the β-β-chain chain gene. This variant causes a mild chronic gene. This variant causes a mild chronic hemolytic anemia.hemolytic anemia.

6.6. Hemoglobin AS - A heterozygous form Hemoglobin AS - A heterozygous form causing Sickle cell trait with one adult gene causing Sickle cell trait with one adult gene and one sickle cell disease geneand one sickle cell disease gene

7.7. Hemoglobin SC disease - A compound Hemoglobin SC disease - A compound heterozygous form with one sickle gene heterozygous form with one sickle gene and another encoding Hemoglobin C.and another encoding Hemoglobin C.

Sickle cell diseaseSickle cell disease Sickle cell disease is a Sickle cell disease is a recessive genetic disorder recessive genetic disorder of of

the blood caused by a the blood caused by a single nucleotide alteration in single nucleotide alteration in the β-globin genethe β-globin gene

This condition is inherited in an autosomal recessive This condition is inherited in an autosomal recessive pattern, which means both copies of the gene in pattern, which means both copies of the gene in each cell have mutations. The parents of an each cell have mutations. The parents of an individual with an autosomal recessive condition individual with an autosomal recessive condition each carry one copy of the mutated geneeach carry one copy of the mutated gene

People with this disorder have atypical hemoglobin People with this disorder have atypical hemoglobin molecules called molecules called hemoglobin Shemoglobin S that is distorted. that is distorted.

A molecule of A molecule of HbS contains two normal α-globin HbS contains two normal α-globin chains and two mutant β-globin chainschains and two mutant β-globin chains, in which , in which glutamateglutamate at position 6 has been replaced with at position 6 has been replaced with valinevaline..

Sickle cell diseaseSickle cell disease Under Under low oxygen low oxygen conditions, the conditions, the absenceabsence of a of a

polar amino acid polar amino acid of the β-globin chain promotes of the β-globin chain promotes the the polymerisation of haemoglobinpolymerisation of haemoglobin, which , which distorts red blood cells into a sickle shapedistorts red blood cells into a sickle shape and and decreasesdecreases their their elasticity.elasticity.

Sickle cells Sickle cells block the flow of blood block the flow of blood in the narrow in the narrow capillaries. capillaries.

This interruption in the supply of oxygen leads to This interruption in the supply of oxygen leads to localized localized anoxiaanoxia (oxygen deprivation) in the (oxygen deprivation) in the tissue, causing pain and eventually death tissue, causing pain and eventually death ((infarctioninfarction) of cells in the vicinity of the ) of cells in the vicinity of the blockage.blockage.

(sickle cell disease video)(sickle cell disease video)

Abnormal HemoglobinAbnormal Hemoglobin

Reasons to suspect a hemoglobin Reasons to suspect a hemoglobin disorder:disorder:– Patient presents with suspicious history Patient presents with suspicious history

or physical examor physical exam– Laboratory tests: Microcytic Laboratory tests: Microcytic

hypochromic RBCs, hemolytic anemiahypochromic RBCs, hemolytic anemia– Screening test abnormality (primarily in Screening test abnormality (primarily in

neonates)neonates)

Most common Hemoglobin Most common Hemoglobin abnormalitiesabnormalities

ThalassemiasThalassemias– AlphaAlpha– BetaBeta

HemoglobinopathiesHemoglobinopathies– HbS trait; diseaseHbS trait; disease– HbC trait; diseaseHbC trait; disease– HbEHbE– Hereditary Persistence of Hemoglobin F Hereditary Persistence of Hemoglobin F

(HPFH)(HPFH)

Laboratory Methods to Laboratory Methods to evaluate Hemoglobinevaluate Hemoglobin

Red cell morphologies:Red cell morphologies: – HbS: Sickle cellsHbS: Sickle cells

Sickle cells on peripheral Sickle cells on peripheral smearsmear


Red cell morphologies:Red cell morphologies:– HbS: Sickle cellsHbS: Sickle cells– HbC: Target cells, crystals after HbC: Target cells, crystals after

splenectomysplenectomy

HbC crystals with Target HbC crystals with Target cellscells


Red cell morphologies:Red cell morphologies:– HbS: Sickle cellsHbS: Sickle cells– HbC: Target cells, crystals after HbC: Target cells, crystals after

splenectomysplenectomy– Thalassemias: Microcystosis, target Thalassemias: Microcystosis, target

cells, basophilic stipplingcells, basophilic stippling

Alpha Thalassemia with Alpha Thalassemia with basophilic stipplingbasophilic stippling


High-Performance Liquid High-Performance Liquid Chromatography (HPLC):Chromatography (HPLC):– Weak cation exchange column. The Weak cation exchange column. The

ionic strength of the eluting solution is ionic strength of the eluting solution is gradually increased and causes the gradually increased and causes the various Hemoglobin molecules to have a various Hemoglobin molecules to have a particular retention time.particular retention time. Amino acid substitutions will alter the Amino acid substitutions will alter the

retention time relative to HbA.retention time relative to HbA. There is some analogy between retention There is some analogy between retention

time and pattern on alkaline electrophoresis.time and pattern on alkaline electrophoresis.

Normal HPLC patternNormal HPLC pattern


Solubility test Solubility test (Sickledex):(Sickledex):– Test to identify HbS. Test to identify HbS.

HbS is relatively HbS is relatively insoluble compared to insoluble compared to other Hemoglobins.other Hemoglobins.

– Add reducing agentAdd reducing agent– HbS will precipitate HbS will precipitate

forming and opaque forming and opaque solution compared with solution compared with the clear pink solution the clear pink solution seen in HbS is not seen in HbS is not present.present.


Electrophoresis:Electrophoresis:– Alkaline (Cellulose Acetate) pH 8.6:Alkaline (Cellulose Acetate) pH 8.6:

All Hemoglobin molecules have a negative charge, All Hemoglobin molecules have a negative charge, and migrate towards the anode proportional to their and migrate towards the anode proportional to their net negative charge.net negative charge.

– Amino acid substitutions in hemoglobin variants alter Amino acid substitutions in hemoglobin variants alter net charge and mobility.net charge and mobility.

– Acid (Citrate agar) pH 6.2:Acid (Citrate agar) pH 6.2: Hemoglobin molecules separate based on charge Hemoglobin molecules separate based on charge

differences differences andand their ability to combine with the agar. their ability to combine with the agar.– Used to differentiate Hemoglobin variants that migrate Used to differentiate Hemoglobin variants that migrate

together on the cellulose gel (i.e. HbS from HbD and together on the cellulose gel (i.e. HbS from HbD and HbG, HbC from HbE).HbG, HbC from HbE).

Hemoglobin Electrophoresis Hemoglobin Electrophoresis PatternsPatterns

Hemoglobin VariantsHemoglobin Variants

Hb A : normal individualHb A : normal individual Hb A + Hb F: normal neonateHb A + Hb F: normal neonate Hb F : beta thalassemia major individualHb F : beta thalassemia major individual Hb S : Homozygous HbS Hb S : Homozygous HbS

individual(individual(α2βα2βS2)S2) Hb A + Hb S: Heterozygous Sickle Hb A + Hb S: Heterozygous Sickle Hb S + Hb C: sickle cell disease Hb S + Hb C: sickle cell disease

individual (individual (α2βα2βC2)C2)

THE ENDTHE END

Hemoglobin Electrophoresis (Biochemistry)

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