Introduction: When amino acids form peptide bonds between the carboxyl terminal and the amino terminal

a protein polymer is formed. These polymers are known as polypeptides and several of these can form an individual protein. Due to the individual chemical and physical properties of each amino acid, the properties of the final protein are determined by the sequence of amino acids (Molecular and Biomedical Sciences, 2012). The physical structure of the protein depends on the hydrophobic or hydrophilic nature of the amino acids. If the R-group side chain is hydrophobic it will be found on the inside of the protein shielded from water. If the R-group is hydrophilic it will be found in the water filled external protein environment. Ionic side chains will also be found in contact with a hydrophilic environment.

Figure 1: The general amino acid structure. The amino acid has an - amino group, an - carboxyl group and a side chain (R) which supplies the protein with its final charge depending on its level of ionization. From: http://www.bioinformatics.org/tutorial/images/aminoacid.gif Each ionisable side chain has a specific pKa value, which is the pH at which the group is half ionised. That is 50% of the ionisable groups are ionised and 50% are not (Molecular and Biomedical Sciences, 2012). For example glutamic acids side chain contains a carboxyl group (COOH). The pKa of this side chain is 4.07, which means when the pH is equal to the pKa 50% of the carboxyl groups are ionised to COO- and 50% remain unionised as COOH. In general basic side chains have a high pKa while acidic side chains have a low pKa. The proteins overall charge is ultimately due to the side chains of the amino acids that make up that specific protein. Therefore the isoelectric point of the protein is determined by the side chains pKa (Molecular and Biomedical Sciences, 2012). The isoelectric point of a protein is the pH at which the protein can exist without an overall charge. The protein will become positively charged as the pH of the environment drops below the pI and negatively charged when the pH of the environment rises higher that the pI (Molecular and Biomedical Sciences, 2012). This phenomena allows us to separate proteins according to their charge. The separation of proteins according to charge is called ion exchange chromatography (Sheehan, 1996). The column is filled with an ion exchange resin, in our case CM cellulose. The CM-cellulose beads act as a weak cation exchanger, exchanging H+ for other positively charged ions. CM-cellulose is

negatively charged and therefore any protein with an overall positive charge will knock off the H+ and bind to the cellulose beads (Molecular and Biomedical Sciences, 2012). Separation due to overall protein charge occurs because proteins with an opposite charge to the cellulose are adsorbed and do not move. Proteins with the same charge or those that are neutral are washed through the resin, as they do not adsorb to the cellulose beads.

Figure 2.0: The negatively charged beads in the column cause positively charged proteins to adsorb to them, while the negatively charged proteins pass through. From: http://en.wikibooks.org/wiki/File:I onexachange.jpg

The overall protein charge can be manipulated by increasing or decreasing the pH of the buffer in the column. As the pH of the buffer increases the proteins overall charge will change. This allows us to separate a protein mixture into its separate components. The aim of the following experiment is to separate a mixture of haemoglobin (pI=6.8) and cytochrome (pI=10.6), using CM-cellulose and a range of Na Phosphate buffers (0.01M pH 6.3, 7.5 and 9.5, and 0.1M pH 9.5 and 10.7). Methods: Please refer to page 70 of the 2013 Practical A Semester 2 workbook. No deviations were made from the protocol. Results: Once the protein mixture (cytochrome C-haemoglobin) had been added to the column a dark brown line was present at the top. At pH 6.3 there was no movement by either protein and the brown line remained on top. Once the buffer pH was increased to 7.5 the brown line began to move through the column very slowly. After 2ml was added the brown line was about a quarter down the column. The 9.5 pH buffer was then added resulting in the red line remaining stationary and the brown line continuing movement down to about halfway through the column. After 1.5ml of 0.1M pH 9.5 buffer had been added the brown band had been completely eluted from the column. The red band continued to remain stationary on top of the column until the pH of the buffer had been increased to 10.7. At this point the red band spread down the column until it was completely eluted after 1.5ml of buffer had been added. Below are tables

summarising the results as described above. The first table shows the overall charge of the protein while the second shows the movement of the band through the column. The two tables can be compared to show which overall charge allows the protein band to begin movement. Table 1.0: Table showing the overall charge of the protein at each buffer pH Protein pI 0.01M 0.01M 0.01M 0.1M pH 0.1M pH pH 6.3 pH 7.5 pH 9.5 9.5 10.7 Haemoglobin (Dark brown) 6.8 Positive Negative Negative Negative Negative Cytochrome C (Red) 10.6 Positive Positive Positive Positive Negative

Table 2.0: Table showing the movement of the protein at each buffer pH Protein pI 0.01M pH 0.01M pH 0.01M pH 0.1M pH 0.1M pH 6.3 7.5 9.5 9.5 10.7 Haemoglobin No Begin Halfway Eluted (Dark 6.8 Movement movement through brown) column Cytochrome C (Red) No No No No Begin 10.6 Movement Movement Movement Movement movement - Eluted

Discussion: The results showed the pH at which the proteins begin to move through the column, more specifically they allow us to determine when the charge of the protein changes from positive to negative. Initially both proteins had an overall positive charge causing them to adsorb strongly to the negative CM-cellulose beads. By adsorbing strongly no movement was observed of either protein. From table 2.0 it was determined that at pH 7.5 the haemoglobin protein began its movement. Movement was initiated because the overall protein charge had changed from positive to negative as can be seen from table 1.0. The overall charge changed the interaction possible between the negatively charged CMcellulose beads and the protein. The haemoglobin was no longer able to adsorb to the beads as strongly due to a decrease in opposite charge attraction and was therefore able to begin passing through the column. As the pH of the buffer increased more amino acid side chains were exposed to a pH higher than their pKa. Due to this more side chains were becoming negatively charged and the overall negative charge of the protein was increasing allowing the haemoglobin protein to decrease its interaction with the negatively charged beads and ultimately move faster. Eventually at pH 9.5 the haemoglobin protein had eluted through the column as can be seen from table 2.0. The table shows two buffers

had a pH of 9.5, however one of these was a stronger concentration (0.1M). By increasing the concentration of the Na phosphate buffer the Na ions are more plentiful and can compete with the proteins for opposite charges on the resin (Molecular and Biomedical Sciences, 2012). By increasing the ionic concentration the protein is further encouraged to pass through the column. The cytochrome C protein had an overall negative charge throughout all the pH ranges except at pH 10.7 (table 1.0). Once pH 10.7 (0.1M) was reached the pH had become higher than the pI causing the protein to adopt an overall negative charge and begin movement (table 2.0). Once again a negative charge allowed the protein to decrease its interaction with the negatively charged CM-cellulose beads and stop adsorbing to their surface. The increase in ionic concentration allowed for competition for binding sites on the resin. These results allow us to draw a conclusion about the different proteins in the protein mixture. It can be seen that as the pH increases haemoglobin begins movement first; this indicates that haemoglobin is a more acidic protein than cytochrome C. It is described above how the overall pI of a protein is ultimately due to the individual pKas of the amino acid side chains. A lower pI indicates the protein contains a higher proportion of low pKa amino acid side chains. Amino acids with low pKa side chains are acidic and therefore it is reasonable to assume that as the haemoglobin began movement at a lower pH than cytochrome C it contained a higher proportion of low pKa amino acid side chains. Allowing the conclusion that haemoglobin is more acidic in nature than cytochrome C. Limitations in the protocol are minimal, however the purity of the cytochrome Chaemoglobin mixture was unknown. It was assumed to be pure, however if contaminated this could have altered results. In addition if the Na phosphate buffer contained contaminants of any kind this may have also altered results (Dionex , 2002). Considering all results are as expected it is reasonable to assume no experimental errors were present, however some may have been detected if the experiment required a more precise data set. Conclusion: It can be concluded that it is possible to separate a mixture of haemoglobin and cytochrome C through ion exchange chromatography. Haemoglobin has a lower pI value and therefore is eluted from the column at a lower pH than cytochrome C. Furthermore the results indicate haemoglobin is more acidic than cytochrome C.

References: Dionex Corporation. (2002) Principles and Troubleshooting Techniques in Ion Chromatography. Available: http://www.iac.tuwien.ac.at/instprakt/ic4.pdf. Last accessed 4th Sep 2013. Molecular and Biomedical Sciences (2012) Practical A (Semester 2) Laboratory Manual. The University of Adelaide, Adelaide, Australia. Osuri, G. (2003). Amino Acids. Available: http://www.bioinformatics.org/tutorial/1-3.html. Last accessed 4th Sep 2013. Sheehan, D. and Fitzgerald R. (1996) Ion-Exchange Chromatography. Methods in Molecular Biology. Volume 59: pp145-150. Wikibooks. (2009). Ion Exchange. Available: http://en.wikibooks.org/wiki/File:Ionexachange.jpg. Last accessed 4th Sep 2013.