The aim of the experiment of this week was the determination of the unknown aminoacid (histidine, arginine, aspartic acid, alanine) by using the pH values obtained whenever a specific amount of the base NaOH was added to plot the titration curve and then to determine the pI value by using this graph.
During this laboratory hour, unknown aminoacid samples had to be identified by using the titration by strong base ( NaOH) and acid (HCl). Initially, the aminoacids were protonated with HCl and afterwards, NaOH was added into aminoacid solution to ensure a buffer system. Due to this procedure, the plot of titration curve for each aminoacid were drawn. The pKa values of aminoacids were demonstrated, where each pKa value is specific for a specific aminoacid. Also, the pI values for each aminoacids were calculated using given pKa values, then they were compared with their theoretical calculations. Due to graph, the first sample was determined to be Aspartic acid due to the acidic activity it shows. The second graph was estimated to be Histidine, while the third and the forth one were determined to be Glycine and Arginine respectively. According to the graph obtained by the data of our experiment, the approximate pI value of Aspartic acid is 7, while the theoretical one found in the database is 5.86. The experimental pI value of Histidine is 3, while the theoretical one is 4.35. The experimental pI value of Glycine is approximately 6, while the theoretical one is 4. The experimental pI value of Arginine according to the graph is approximately 5.5, while the theoretical one 6.05. The calculated pI value found by using the pKa values of the ionizable groups of the hexapeptide “DENISA” is 3.15, while the one found in the database is 3.67. The calculated pI value was found by using the pKa values of the ionizable groups of the pentapeptide “NAYCI” is 5.345, while theoretical one obtained from the database is 5.52. The calculated pI value found by using the pKa values of the ionizable groups of the tetrapeptide”RHAN” is 10.735. while the theoretical one obtained from the database is 9.76 . The reasons why there is a mismatch between the theoretical and the experimental pI value can be because of some of the side chains which can be buried or located in salt bridges. The environment where the aminoacids are located affects the aminoacids leading therefore to changes in pKa and in the pI value as well. Additionally the presence of the ionic lipid headgroups near the aminoacids lead to a change in the pKa values due to the ionic activity . The changes in pH during titration can affect the ionizable groups and this can also be the reason about the changes in each specific pKA and the pI value as well. Also it had to be mentioned that the values found in the database are calculated in the optimum conditions .
1.http://www.laney.edu/wp/cheli-fossum/files/2012/01/Amino-Acid-Sidechains.pdf retrieved 01.11.2016 from Internet source
2.http://web.expasy.org/cgi-bin/compute_pi/pi_tool retrieved 01.11.2016 from Internet source
3.https://www.researchgate.net/post/What_is_the_significance_of_the_theoretical_pI_of_a_protein_calculated_using_bioinformatics_tools_when_it_is_expressed_in_the_body retrieved 01.11.2016 from Internet resource
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911520/retrieved 01.11.2016 from Internet resource