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Separation by Paper Electrophoresis

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This essay investigates the literature available on paper electrophoresis. This is the main separation technique that is usually applied biological laboratories. The operation of this technique is based on the scientific fact that molecules separate depending on the charge distribution that is characteristic of the molecules being separated. Ideally, separation is achieved regardless of the medium used or the actual physical arrangement of the apparatus. In most cases, the support medium is composed of gel contained within a narrow tube or in flat sheets. The common detergent used for this experiment is sodium dodecyl sulphate. However, this detergent mainly applies for bio-molecules of protein nature. According to literature, electrophoresis has become the ideal method of determining molecular weights. In electrophoresis of amino acids, the differential electrical distribution in various types of proteinss was used to elucidate the actual biochemical difference in their structures. Accordingly, the amino acids migrated at a rate different from that of others with a different charge distribution. This clearly showed that the various types of amino acids have quite different biochemical structures (DeWayne, 1993).

Electrophoresis exploits the fact that amino acids exist as zwitterions in various pH values of the solutions in which they are dissolved due to the existence of both a carboxylic acid group and a basic amino group. As the pH of the solution changes, there occurs an internal transfer of hydrogen ions from the carboxylic acid group to the amino group. This leads to a situation where the carboxylic acid has a negative charge while amino group has a positive charge. In biochemical terms, this is termed as Zwitterion. Ideally, this is the predominant form in which most amino acids exist while in solid state or in a simple aqueous solution (Reighton, 1993).

A zwitterion can have an overall charge of positive or negative depending on the number and nature of the charged groups present in the structure. This often happens such that when the pH of the solution is raised by adding hydroxide ions to it, hydrogen ion escapes from the amino group to obtain a negative charge. This can ideally be shown using the technique of electrophoresis (DeWayne, 1993).

  1. Make a rough drawing/photograph of your electrophoretogram (3 marks)
  1. Tabulate the colour of each spot and the direction (+ towards anode/- towards cathode) and the distance (mm) it has travelled from the origin. Immediately obtain results from other groups for the two buffers you did not use. This will allow you to complete the table (below). (6 marks).

Amino acid

pH

Colour

Direction (+/-)

Distance (mm)

Asp

6.0

Purple

+

1/2

Leu

6.0

Purple

-

1/2

Lys

6.0

Purple

-

1

What is the probable composition of the unknown mixture? Explain your reasoning? (6 marks)

The unknown mixture is likely to be made up of Aspertate and Lysine. This basically stems from the fact that the control experiment produced a purple spot of 1 cm towards the anode and 0.5 cm towards the cathode. Moreover, there was no spot left at the point of introduction of the samples ruling out any possibility that Leuine, without a charge, remained stationary. According to literature, Aspertate has an overall positive charge while Lysine has an overall negative charge in buffer systems whose pH values are pH3 and pH10 respectively. Conversely, Leucine would have no overall charge at pH value of pH 6.1. These relative charges will determine the distance moved in the electrophoregram.

  1. By reference to the structures of the three amino acids, and using their pK values (below), calculate the approximate net charge of the amino acids at the different pH value.

Amino acid

pKA (COOH)

pKB (NH2)

pKR (R-group)

Asp

2.0

9.9

3.9

Leu

2.3

9.7

N/A

Lys

2.2

9.2

10.8

If pH > pK then an ionisable group loses a proton.

Complete and fill in the table (below) showing the approximate net charge of the amino acids at each pH (3,6.1,10) value (12 marks).

pH

Amino Acid

*COOH

*NH2

*R-Group

Overall Charge

3

Asp

+

-

-

-

3

Leu

+

-

N/A

0

3

Lys

+

+

-

+

6.1

Etc.

 

 

 

 

* write the formula of the ionisable groups at each pH (COOH/COO-  NH2/NH3+) this will allow you to calculate the overall charge on each amino acid at each pH.

  1. Compare your experimental results with your calculated charges and comment on these comparisons (6 marks).

The calculated values conform to the experimental findings. For instance, the sample solution containing leucine, lysine and aspertate amino acids produced three distinct spots. The spots were distributed to both sides of the baseline while one was retained on the line. This certainly agrees with the scientific fact that lysine, bearing an overall positive charge, will migrate towards the anode, while aspartic acid with an overall charge of negative will migrate towards the cathode. Moreover, leucine with a zero overall charge would not migrate from the baseline.

  1. Why were you instructed to handle the paper at the ends only? (4 marks)

This was to ensure that our hands do not contaminate the paper by introducing amino acids that were not present in the original sample. It basically ensures accuracy of the results. By handling at the ends, the region of the paper that is supposed to be used for the electrophoretic experiment will not be disturbed. For instance, the migrating individual amino acids will limit themselves to the central region of the paper, thereby, eliminate any possible errors introduced by human contamination.

  1. Briefly discuss the use of electrophoretic separations in the determination of Sickle Cell Anaemia. Make sure you explain the underlying biochemistry of the separation process (10 marks).

Electrophoretic separation of sickle cells from normal red blood cells applies the principle of enzyme specificity. For instance, the normal gene haemoglobin can be digested enzymatically into smaller particles. When these fragments are put through electrophoretic separation, the experimental probe is observed to be bound to smaller the small fragments. However, sickle cells cannot be digested by the enzymes, thus, the experimental probe binds to larger fragments.

According to the experimental findings, sickle cells migrated at a rate different from that of normal red blood cells. This clearly shows that the two types of cells have quite different biochemical structures, considering that electrophoresis separation are based on electric charges. In fact, the results indicate that sickle cell trait could easily be obtained by mixing normal cells and the abnormal sickle cells (Reighton, 1993).

The underlying biochemistry that is exploited in haemoglobin electrophoresis is the fact that the amino acid Valine replaces Glutamic acid that is present in normal haemoglobin. Sickle cell anaemia is a typical genetic disorder which is cgaracterized by genetic coding of Valine instead of Glutamic acid at the sixth position of the beta chain of the hemoglobulin molecules. At the genetic level, the only difference exists in the substitution of Thymine for Adenine at the middle position of codon number six (DeWayne, 1993).

Conclusion

In conclusion, electrophoresis of amino acids is one of the simplest forms of electrophoretic determination. Although amino acids have no distinct colours that can be used to identify them, a purple colour or yellow colour can be obtained by simply spraying the experimental paper with ninhydrin solution. In most cases, the paper is then allowed to dry before gentle heating to have the spots showing up.

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