Sickle Cell Anaemia- A Molecular Disease of Haemoglobin :

Haemoglobin is found in the erythrocytes (red blood corpuscles) of chordate animals. It is such a macromolecule that transports oxygen from lungs to all tissues of body.

Each molecule of haemoglobin consists of a haeme group plus four polypeptide chains e.g. alpha, beta or gamma. Each alpha polypeptide chain is made up of 141 amino acids, while the beta and gamma chains consist of 146 amino acids.

What is Sickle Cell Anaemia?

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There are over 4000 genetic diseases occurring in human population. Most of these variants differ in single amino acid substitution; a Val instead of Glu residues is present at position 6 in two beta chains. This replacement results in ‘sticky’ hydrophobic contact points at position 6 of beta chain.

These sticky points cause deoxyhaemoglobins to dissociate abnormally. Sickle cell anaemia arises due to mutation. Sickle cell haemoglobin (i.e. haemoglobin) is such a variant. Haemoglobin molecules precipitate upon deoxygenation, and form crystal like aggregates which distort the morphology of RBCs. The RBCs elongate to form sickle-shaped cells.

Here the word ‘sickle’ refers to an agricultural tool used for cutting of crop plants. Sickled cells are very fragile and rupture easily resulting in gradual loss of blood. This situation is called ‘anemia’.

Blood capillaries get blocked resulting in death of children, if not given regular medical treatment. The sickled RBCs re severe in malaria-prevalent areas such as India

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Protein Fingerprinting (Peptide Mapping) of Hemoglobin:

V.A. Ingram (1957) first confirmed differences between normal haemoglobin and haemoglobins. Since then this technique is being used to compare two or more proteins obtained from different sources.

Protein fingerprinting of normal and sickled haemoglobin is accomplished in the following steps.

1. First procure pure haemoglobin from normal RBC and sickle-cell RBC and separately transfer in a test tube

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2. Transfer trypsin to digest them as it cleaves the peptide at peptide bond site near lysine or arginine residue.

3. Spot trypsin-digest protein on Whatman filter paper and pass through electrophoresis at pH 2.1.

4. Take out the filter paper strips and dry them.

5. Carryout paper chromatography using the mixture of butanol: acetic acid : water (4:1:5) (peptides separate on the basis on hydrophobicity of hydrophilicity. Generally hydrophobic peptides move faster than hydrophilic ones).

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6. Take out the paper strips and dry them. Spray ninhydrin solution on the paper strips (all the peptides of both normal and sickled haemoglobin are normal except one, as marked).

The different (non-overlapping) peptides were eluted from paper and sequenced. It was found that at position 6 from -NH2 terminus, glutamate (normal haemoglobin) was replaced by valine (in haemoglobin).

This substitution event results in increased hydrophobic interaction in haemoglobin molecules. That is why it causes deformity in RBCs (to B chain) which appears sickle shaped.

Protein fingerprinting technique is applicable in comparing many proteins isolated from various sources. Use of computer has helped a lot in searching out homology of proteins. This work has given birth to a new era of biology plus computer science and called ‘Bioinformatics’.