Isolation of lysozyme from egg white. 2 PDF

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Introduction The aim of this experiment was to purify lysozyme from egg white using ion – exchange chromatography. An SDS page gel was also carried out to monitor the purification of through assay of lysozyme activity. It is important to be able to use these methods of purification as Lysozyme has an important antibacterial role in egg white, that is considered in the discussion below. Thus, these methods are crucial in scientific theory. Ion – exchange chromatography exploits variances in the sign and magnitude of the net electric charges of the proteins at a given pH (Nelson, 2013). The column works because it contains charged groups. The protein binding affinity to the charged groups in the column depends on the pH as that is what determines the ionisation state of the molecule. There can be both charged groups bound with anionic groups, cation exchangers, and some bound with cationic groups, anion exchangers (Nelson, 2013). The mobile phase is a protein solution and the expansion of this is caused by the separation of proteins with different properties. Ion exchange chromatography is useful as it is one of the only techniques that is able to separate difficult molecules that aren’t as easily separated with other techniques (Bio-Rad, 2016). The sample, that was being extrapolated in this experiment was pre-treated egg white with a minimum of nine varying proteins’. The resin in the column was a cation exchanger, CM-Sephadex, positively charged ions were binding to the negatively charged resin. The experiment also contained three varying buffers that increased in pH these were; Acetate pH 4.6, Phosphate buffer pH 7.0 and Carbonate buffer pH 11.4. The way in which a protein is purified by ion exchange chromatography is mainly due to selected conditions. Using the right conditions means the contaminates will bind to the resin in the column, during both the binding and elution stages of the buffer, as the protein passes straight through the column. A spectrophotometer was used to detect the protein concentration after ion exchange chromatography had taken place. The basic principle of how a spectrophotometer works is down to its ability to measure the intensity of light/photons absorbed by an object. Protein in solution absorbs UV light at around 280 nm, with peptide bonds giving readings of about 200nm and aromatic rings giving readings at 280nm. In the second half of the experiment a sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) was carried out. This is a popular method for protein separation. Proteins migrate toward the negative electrode during the electrophoresis. The proteins are parted based on their molecular weight (Oswald, 2016). However, it is not as straightforward as proteins are folded numerous times and their net charge nor molecular radius is dependent on their overall weight. The

molecular radius of a protein is determined by the tertiary structure. In order to allow the separation of the protein by just molecular weight, we need to break down the tertiary structures to make it linear. SDS is used in this procedure to denature the proteins, therefore making it linear. In addition to denaturing the protein, SDS is useful in this technique as it masks the protein in a uniform negative charge effectively removing any interference, coming from the R groups on the amino acids, that could have otherwise disrupted the results of the electrophoresis (Oswald, 2016). The addition of SDS to the gel is to make sure these changes to the protein continue throughout the experiment. The determining factor hence has become molecular weight as the proteins have the same charge to mass ratio and the molecular radii is determined by the molecular weight. The gel used for SDS-PAGE is polyacrylamide as it is chemically inert. The protein can then run through the buffer with the lower weighted molecules moving much more hastily through the gel.

Method Please refer to protocol. Results Fraction Number

A280 Acetate Buffer

1 2 3 4 5 6 7 8

2.5 0.9 0.3 -0.002 -0.14 -0.16 -0.17 -0.2

– Fraction Number

9 10 11 12 13 14 15 16

A280 – Fractio Phosphate n Buffer Numbe r -0.002 17 0.12 18 1.0 19 1.3 20 0.4 21 0.2 22 0.3 23 0.2 24

A280 – Carbonate Buffer 0.5 0.2 0.1 0.1 0.02 0.07 0.1 0.1

There are quite a few minus results for the Acetate Buffer which was not expected. The data point of the Phosphate and Carbonate buffer seem to be the most similar.

Graph of fraction number vs absorbanc e 3

Absorbance 280

2.5 2

pH 7.0

pH 4.6

pH 11.4

1.5 1 0.5 0 0

5

10

15

20

25

30

-0.5 FRaction number

The graph shows the unexpected negative absorbance’s that was mentioned above. After the Acetate buffer, the data starts to become more probable with slight variations but positive absorbance. The Phosphate and Carbonate buffers seem to share the most similar data out of the three. Suggesting these two buffers are better suited for this purification. Discussion From the above experiment, we know that the egg white contained Lysozyme. The reason it does is that Lysozyme acts as an extracellular enzyme in egg whites catalysing the hydrolysis of β linkage between Nacetylmuramic acid (NAM) and N-acetyl-glucosamine (NAG) subunits in the peptidoglycan polymers (Nicely, 2006). This hydrolysis leads to the breakdown of polymers that compose bacterial cell walls. Lysozyme can act as an enzyme as it had two subdomains that can bind to saccharide substrate (Nicely, 2006). The results of this experiment only show one-half of the practical and so the first half is what this section will cover. The main result from this practical is the absorbance that was detected by the spectrophotometer for each buffer. An important point to mention is the isoelectric point of Lysozyme which is 11.0 (Wetter, 1951). An isoelectric point is a pH at which a molecule has no net charge. The first obvious characteristic of the graphs above is for the Acetate buffer there are lots of negative absorbance readings. This is probably due to a physical issue rather than a problem with the solution, as in it is possible the cuvettes used weren’t filled high enough in order for the light to diffract through the solution effectively enough to produce an accurate reading. This is down solely to human error and to make the experiment

more reliable in the future it would be important to put the correct amount of solution in each cuvette. The peak of this section of the graph shows a rapid decrease in absorbance for this buffer, this, however, could be due to the lack of solution in the cuvette as well – the experiment may have started with the correct amount of solution but ended with the incorrect amount. There is a sharp increase in absorbance with the Phosphate buffer. Again as with the Acetate buffer the first absorbance that was read from the spectrophotometer was negative and probably due to experimental error. The Carbonate buffer has the most data points that are close together. This consistency gives the impression that the results produced by this buffer have the least amount of contaminants in the product. Molecules change pH based on their environment, above their pI, they carry a negative charge. The isoelectric point for Lysozyme is 11.0 (Wetter, 1951) and the Carbonate buffer had a pH of 11.4 slightly above the pI of Lysozyme. This supports the data above, as we used a cation exchanger the resin was also negative hence the Lysozyme would not have been binding to the resin and would have passed through the column, whilst the containments would have been bound to the resin. This also supports the results for the Phosphate buffer as the opposite occurred. Since the buffer was lower in pH than the isoelectric point for Lysozyme the Lysozyme would have been positive meaning it could have been bound to the resin. This also explains the sharp peak for the Phosphate buffer data. Some containment may have got through with the Lysozyme. The A280 method for protein concentrations is not always the most reliable as other containments absorb at that wavelength like nucleic acids. The second half of the experiment does contain a further analysis of these results making the whole experiment more accurate. If, however, there was a need to further solidify the accuracy and reliability of these results there are other methods of protein analysis that could have been carried out. Such as Bradford or Lowry assays. Another chromatographic technique that could have been carried out is size exclusion chromatography which quite simply put is exactly as it says. A technique used to separate molecules based on their size through a gel (Heidenreich, 2011). This technique is also widely used for protein concentration determination. References Bio-Rad Laboratories. (2016). Retrieved on 8/12/2016 http://www.bio-rad.com/en-uk/applications-technologies/liquidchromatography-principles/ion-exchange-chromatography

David L. Nelson, Michael M. Cox. (2013). Lehninger Principles of Biochemistry. (Sixth Edition). New York: W.H Freeman and Company

Wetter, L.R., et al. (1951). J. Biol. Chem. 192, 237-242

Nicely, N. (2006). Study of Protein Binding Sites on the GTPase RalA and the Sugar-binding Protein Hen Egg White Lysozyme. 1

Heidenreich, A.J. (2011). Synthesis and Characterization of Arborescent (Dendritic) Polystyrenes Prepared by Raft Polymerization. 63-64...