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Introduction:When amino acids form peptide bonds between the carboxyl terminal and theamino terminal a protein polymer is formed. These polymers are known aspolypeptides and several of these can form an individual protein. Due to theindividual chemical and physical properties of each amino acid, the properties ofthe final protein are determined by the sequence of amino acids (Molecular andBiomedical Sciences, 2012). The physical structure of the protein depends on thehydrophobic or hydrophilic nature of the amino acids. If the R-group side chainis 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 proteinenvironment. Ionic side chains will also be found in contact with a hydrophilicenvironment.

Figure 1: The general amino acid structure. The amino acid has an - aminogroup, an - carboxyl group and a side chain (R) which supplies the protein withits final charge depending on its level of ionization.

From: http://www.bioinformatics.org/tutorial/images/aminoacid.gif

Each ionisable side chain has a specific pK a value, which is the pH at which thegroup is half ionised. That is 50% of the ionisable groups are ionised and 50%are not (Molecular and Biomedical Sciences, 2012) . For example glutamic acidsside chain contains a carboxyl group (COOH). The pK a of this side chain is 4.07,which means when the pH is equal to the pK a 50% of the carboxyl groups areionised to COO - and 50% remain unionised as COOH. In general basic side chainshave a high pK a while acidic side chains have a low pK a.

The proteins overall charge is ultimately due to the side chains of the aminoacids that make up that specific protein. Therefore the isoelectric point of theprotein is determined by the side chains pK a (Molecular and Biomedical Sciences,2012). The isoelectric point of a protein is the pH at which the protein can existwithout an overall charge. The protein will become positively charged as the pHof the environment drops below the pI and negatively charged when the pH ofthe 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 exchangechromatography (Sheehan, 1996). The column is filled with an ion exchange

resin, in our case CM cellulose. The CM-cellulose beads act as a weak cationexchanger, exchanging H + for other positively charged ions. CM-cellulose is

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negatively charged and therefore any protein with an overall positive charge willknock off the H+ and bind to the cellulose beads (Molecular and BiomedicalSciences, 2012). Separation due to overall protein charge occurs becauseproteins 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 theresin, as they do not adsorb to the cellulose beads.

The overall protein charge can be manipulated by increasing or decreasing thepH of the buffer in the column. As the pH of the buffer increases the proteinsoverall charge will change. This allows us to separate a protein mixture into itsseparate components. The aim of the following experiment is to separate amixture of haemoglobin (pI=6.8) and cytochrome (pI=10.6), using CM-celluloseand a range of Na Phosphate buffers (0.01M pH 6.3, 7.5 and 9.5, and 0.1M pH 9.5and 10.7).

Methods:Please refer to page 70 of the 2013 Practical A Semester 2 workbook. Nodeviations were made from the protocol.

Results:Once the protein mixture (cytochrome C-haemoglobin) had been added to thecolumn a dark brown line was present at the top. At pH 6.3 there was nomovement by either protein and the brown line remained on top. Once the bufferpH was increased to 7.5 the brown line began to move through the column veryslowly. After 2ml was added the brown line was about a quarter down thecolumn. The 9.5 pH buffer was then added resulting in the red line remainingstationary and the brown line continuing movement down to about halfwaythrough the column. After 1.5ml of 0.1M pH 9.5 buffer had been added the brownband had been completely eluted from the column. The red band continued toremain stationary on top of the column until the pH of the buffer had beenincreased to 10.7. At this point the red band spread down the column until it wascompletely eluted after 1.5ml of buffer had been added. Below are tables

Figure 2.0: The negatively chargedbeads in the column causepositively charged proteins toadsorb to them, while thenegatively charged proteins passthrough.From:http://en.wikibooks.org/wiki/File:Ionexachange.jpg

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summarising the results as described above. The first table shows the overallcharge of the protein while the second shows the movement of the band throughthe column. The two tables can be compared to show which overall chargeallows the protein band to begin movement.

Table 1.0: Table showing the overall charge of the protein at each buffer pHProtein pI 0.01M

pH 6.30.01MpH 7.5

0.01MpH 9.5

0.1M pH9.5

0.1M pH10.7

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 pHProtein pI 0.01M pH

6.30.01M pH7.5

0.01M pH9.5

0.1M pH9.5

0.1M pH10.7

Haemoglobin(Darkbrown)

6.8No

MovementBegin

movementHalfwaythroughcolumn

Eluted-

CytochromeC (Red) 10.6

NoMovement

NoMovement

NoMovement

NoMovement

Beginmovement- Eluted

Discussion:The results showed the pH at which the proteins begin to move through thecolumn, more specifically they allow us to determine when the charge of theprotein changes from positive to negative. Initially both proteins had an overallpositive charge causing them to adsorb strongly to the negative CM-cellulosebeads. 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 itsmovement. Movement was initiated because the overall protein charge hadchanged from positive to negative as can be seen from table 1.0. The overallcharge changed the interaction possible between the negatively charged CM-cellulose beads and the protein. The haemoglobin was no longer able to adsorbto the beads as strongly due to a decrease in opposite charge attraction and wastherefore able to begin passing through the column. As the pH of the bufferincreased more amino acid side chains were exposed to a pH higher than theirpKa. Due to this more side chains were becoming negatively charged and theoverall negative charge of the protein was increasing allowing the haemoglobinprotein to decrease its interaction with the negatively charged beads andultimately move faster. Eventually at pH 9.5 the haemoglobin protein had elutedthrough the column as can be seen from table 2.0. The table shows two buffers

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had a pH of 9.5, however one of these was a stronger concentration (0.1M). Byincreasing the concentration of the Na phosphate buffer the Na ions are moreplentiful and can compete with the proteins for opposite charges on the resin(Molecular and Biomedical Sciences, 2012). By increasing the ionicconcentration the protein is further encouraged to pass through the column. Thecytochrome C protein had an overall negative charge throughout all the pHranges except at pH 10.7 (table 1.0). Once pH 10.7 (0.1M) was reached the pHhad become higher than the pI causing the protein to adopt an overall negativecharge and begin movement (table 2.0). Once again a negative charge allowedthe protein to decrease its interaction with the negatively charged CM-cellulosebeads and stop adsorbing to their surface. The increase in ionic concentrationallowed for competition for binding sites on the resin.

These results allow us to draw a conclusion about the different proteins in theprotein mixture. It can be seen that as the pH increases haemoglobin beginsmovement first; this indicates that haemoglobin is a more acidic protein thancytochrome C. It is described above how the overall pI of a protein is ultimatelydue to the individual pK as of the amino acid side chains. A lower pI indicates theprotein contains a higher proportion of low pK a amino acid side chains. Aminoacids with low pK a side chains are acidic and therefore it is reasonable to assumethat as the haemoglobin began movement at a lower pH than cytochrome C itcontained a higher proportion of low pK a amino acid side chains. Allowing theconclusion that haemoglobin is more acidic in nature than cytochrome C.

Limitations in the protocol are minimal, however the purity of the cytochrome C-haemoglobin mixture was unknown. It was assumed to be pure, however if

contaminated this could have altered results. In addition if the Na phosphatebuffer contained contaminants of any kind this may have also altered results(Dionex , 2002).

Considering all results are as expected it is reasonable to assume noexperimental errors were present, however some may have been detected if theexperiment required a more precise data set.

Conclusion:It can be concluded that it is possible to separate a mixture of haemoglobin andcytochrome C through ion exchange chromatography. Haemoglobin has a lowerpI value and therefore is eluted from the column at a lower pH than cytochromeC. Furthermore the results indicate haemoglobin is more acidic than cytochromeC.

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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 Sep2013.

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 4 th Sep2013.

http://www.iac.tuwien.ac.at/instprakt/ic4.pdfhttp://www.iac.tuwien.ac.at/instprakt/ic4.pdfhttp://www.bioinformatics.org/tutorial/1-3.htmlhttp://www.bioinformatics.org/tutorial/1-3.htmlhttp://en.wikibooks.org/wiki/File:Ionexachange.jpghttp://en.wikibooks.org/wiki/File:Ionexachange.jpghttp://en.wikibooks.org/wiki/File:Ionexachange.jpghttp://www.bioinformatics.org/tutorial/1-3.htmlhttp://www.iac.tuwien.ac.at/instprakt/ic4.pdf