Question 1.Protein Purification
(a) The peptide Lys-Gly-Ala-Glu is stored in a buffer of pH 6.0. Using chemical reasoning predict the migration direction (anodal, cathode or stationary) of the peptide at pH 6.0 based on your understanding of the nature of the constituent amino acids in the tetra peptide.
Figure 1.
In electrophoresis, amino acids that are negatively charged migrate toward the positively charged anode and vice versa. This means that amino acids move towards the anode at a high pH value and move toward the cathode at a lower pH value. The amino acids would not move or migrate when the pH value of the solution within which the peptide is stored is adjusted to the iso-electric point of the amino acid. This affirmation has an implication that since the peptide was stored in a solution with a pH of 6.0, there will be no movement in this solution and thus, the amino acids will remain stationary and intact within the peptide.
(b) If you had a protein X, which was a soluble enzyme found inside the peroxisome, and you wished to separate it from a similar protein Y which was found imbedded in the mitochondrial membrane what would your initial techniques be for isolating these proteins?
NOTE; The subcellular organelles Golgi apparatus ρ = 1 11 g/ cm3, mitochondria ρ= 1.19 g/ cm3 and peroxisomes ρ = 1.23 g/cm3 have ρ as the density of the particles.
The precipitation method is the most suitable to be used in this case due to the solubility of the proteins under analysis. The initial technique will be to add ammonium sulphate to the mixture of proteins X and Y so as to attain a conventional saturation rate of 40%. The ammonium sulphate is added gradually and gently stirred to avoid surface denaturation. The precipitated proteins will be remove by centrifugation and then resuspended continuously so as to ensure that all the proteins that are soluble are isolated from the insoluble protein particles. The first proteins to be purified are water-soluble proteins, isolation of the mitochondria from the cells before purifying a protein located in the mitochondrion’s membrane.
(c) In an ultra centrifuge experiment a protein lactalbumin is dissolved in a pH 6.8, 0.10 M phosphate buffer and held at 28° C. The initial distance of the protein boundary from the axis was 5.949 cm at the start of the experiment (see Figure 2 “a” below) and 6.731 cm when the boundary was measured in the cell 1 hour and 10 minutes later (see Figure 2 “b” below). If the speed of the rotor was 54,000 rpm calculate the sedimentation coefficient s (sec).
Figure 2.
Then the sedimentation coefficient s (sec) can be calculated by the following equation
1 x 2.303/{(2 x 3.142 x 54,000)/60 }x log[5.949/6.731] = 0.2
(d) A direct current was passed through a solution containing valine, histidine and aspartic acid at a pH 6.00. One amino acid migrated to the positive electrode, one migrated to the negative electrode and one did not migrate to either electrode. With careful chemical reasoning using the nature of the amino acid mixture predict which amino acid went where.
The migration or movements of the amino acids in the mixture were underpinned by the variations in their pH values. Amino acids with a higher pH value migrate to the anode while those with a lower pH value move towards the cathode. There are instances when some of the amino acids do not move due to the solution having an equal pH value as theirs; a state called the isoelectric pH value. These properties are suitable in the purification and separation of proteins in their mixtures through a process called electrophoresis.
