Size Exclusion chromatography is a useful method for separating molecules according their sizes and shapes. Stationary phase consists of beads with pores. Large molecules that has great volume than pore size can be excluded from solvent. Smaller molecules has to pass through those beads, this way smaller molecules obtained at last. Sample has to be dissolved in a liquid or gaseous fluid that called as mobile phase.Stationary phase consist of porous matrix to retard molecules by some properties.In the size exclusion method this matrix consist of porous structure with no binding affinity, and separate molecules by their size. Sample has to interact with these phases to purifying by their size through column. Eluent, has analyte, would be obtained this way. In this section stationary phase was Sephadex G75 that seperates molecules that has molecular weight between 3.000- 80.000 Da .(1*)
Materials and Methods:
200 mM NaCI 100mM Phospate buffer with pH 7 was prepared.Chromatography coloumn was prepared with bead powder G75. 80 μl sample was loaded with blue dextran and orange G.Loaded side were covered with piece of coton and 1 volume elution buffer added through experiment. When blue dextran reached to bottom of the column fractions were collected. 4 droplets for each tube was collected in this step. After orange G were obtained few more tube was collected, then collecting procedure was stopped.
Table 1. shows log MW and Ve/Vo values of standard samples and MBG 311’s Ve/Vo value.
Molecular weight calculation of MBG311 :
X=1.460722 when it placed in to equation
Y=1.765 that is logMW
101.765= 58.2 molecular weight.
Blue dextran was obtained first, because it has higher molecular weight than others.It can excluded from porous structure so it can reach faster than others.Oval albumin would be obtained before than lysosyzme because it has a greater molecular weight than lysosyzme. Oval albumin has 42.7 kDa and lysosyzme has 14.388 kDa molecular weight. Orange obatined last because it has smaller molecular and has to loss time when pass through bead’ porous structure of gel. Size exclusion one of the purifying methods for proteins. Other procedures can be applied for purification, like ion exchange,salting out or affinity chromatography . When applying ion exchange or salting out , generally obtained proteins not specific as size exclusion, because similarity between ionic forms and salt concentration not enough to seperate similar protein. For higher specific result size exclusion could be chosed in among them.However, affinity chromatography gives higher specific results and pure protein of interest. If specifity is important, affinity chromatography could be chosen rather than size exclusion. When higher amount of protein is needed an purity has second place than size exclusion could be chosen.2*
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