Clinical Chemistry Lesson 3 Part 4: Eletrophoresis Part 3 Hemoglobin Electrophoresis and Hemoglobinopathies

Hemoglobin

Is a major constituent of the erythrocyte and is a protein that is responsible for the transport of oxygen throughout the circulation

2 alpha chains and 2 beta chains

Adult hemoglobin consists of four polypeptide chains, what are these?

porphyrin ring with an iron atom in its center

➤ formation of alpha and beta chains is regulated by separate genes; each type of chain is synthesized independently of the other type

➤ each chain also contains a __________ _________. The iron is the site of binding for oxygen

True

As 02 attaches to the porphyrin, the three dimensional shape of the CHON changes, the more 02 which attaches, the more the CHON conformation is altered. when 02 leaves the Hgb molecule, the shape returns to its original configuration True or False?

1. Hemoglobin A

2. Hemoglobin A2

3. Hemoglobin F

THREE FRACTIONS OF HGB FOUND IN THE NORMAL RBC

Hemoglobin A

Composed of two alpha and two beta chains.

95%

Hemoglobin A % component

Hemoglobin A2

Consists of two alpha and two delta chains

3.5% of the total

Hemoglobin A2 % component

Hemoglobin F

Consist of two alpha and two gamma chains

2%

Hemoglobin F % component

HEMOGLOBIN ELECTROPHORESIS

➤ first step is removal of Hgb from the erythrocytes

➤ blood is collected with an anticoagulant and centrifuged to separate RBC from plasma

➤ plasma is discarded and the RBC is washed once with 0.155m NaCl and centrifuged again

➤ distilled H2O (1:9 ratio of cells to H2O) is then added to the cellular precipitate to lyse the RBC

➤ centrifugation removes the stroma (RBC wall), leaving the hemolysate ready for electrophoresis

➤ fractionation of Hgb takes place at ph 8.6 on either CA or Agarose

➤ either Ponceau S or Amido black may be used for staining after the separation is complete

➤ since fractionation at ph 8.6 does not always reveal every abnormal fraction, a second separation at ph 6.2 on citrate agar is strongly recommended to obtain further data

HEMOGLOBIN ELECTROPHORETIC PATTERNS

➤ at ph 8.6; Hgb A migrates the further toward the anode, Hgb F is found somewhat behind Hgb A but Hgb A2 does not migrate very far from the origin

➤ several abnormal Hgb fraction (incl Hgb S) are found between Hgb A2 and Hgb F

➤ Hgb C, Hgb D, Hgb E travel the same distance as Hgb S and cannot be fractionated at this ph

SICKLE CELL DISORDER

Abnormal shape of red blood cells that cannot carry as much oxygen

The presence of Hgb S in the RBC produces this type of disorder

Hgb S

is an abnormal Hgb in which the sixth amino acid in the beta chain is valine instead of glutamic acid

sickle cell trait and sickle cell disease

two forms of sickle cell disorder

sickle cell trait

heterozygous for the disorder, inherited the Hgb S gene from one parent and the normal Hgb a gene from the other

sickle cell trait

after electrophoresis this shows an approximately 35% Hgb S and the remainder the normal Hgb A

sickle cell disease

involves inheritance of Hgb S genes from both parents (homozygous disorder)

sickle cell disease

after electrophoresis this show no Hgb A only Hgb S

THALASSEMIA

➤ comprise a series of Hemoglobin disorders in which one of the normal subunit is synthesized only in extremely low concentrations or is not produced at all

➤ the overall clinical effect of these disorders is a greatly diminished formation of red cells, with all the complications of severe anemia

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09/14/2025

09/14/2005