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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.
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).
* 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.
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.
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.
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).
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.