INVESTIGATION OF THE SEPARATION OF PROTEINS

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The main reason for protein purification is to isolate a protein of interest from the rest of the contaminants, so its conformation (structure) and other figures can be studied. This has always been fundamental to many biochemical experiments. Once require region of cellular protein is established, the protein is then liberated into solution and separated from contaminating materials. This is done by sequential use of different fraction techniques. In order purify proteins; one or more of the separation techniques has to be exploited. The solubility of the protein, its size, charge, hydrophobicity or specific binding affinity and its preferential binding was determined. In this experiment, the following steps were followed. Ion exchange chromatography, where proteins are separated base on their charge differences. This was used to elute fractions of proteins and using spectrophotometer to measure the absorbance. Fractions containing peak absorbance (as shown in graph 1) where used in the SDS-PAGE gel electrophoresis. In SDS-PAGE, proteins were separated base on their molecular weight. This shows how pure the purification is by corresponding the distance migrated by protein molecules against the molecular masses of the standard proteins as shown on graph 2.

Bovine serum albumin is a key protein factor of bovine plasma (5g/100mL) as it total up to about 40% of the total body pool of albumin, with the rest found in bodily secretions and interstitial spaces. BSA serves as a delivery machine in vivo and is made in the liver. Its wider use in the range of applications is due to its reversibility binding capacity to ligands. BSA is used as a stabilizing driving force in enzymatic reactions and as a transporter protein in several vaccines and medicines. There has been increased awareness given to the use of BSA in medications of late, due to its potential contaminations by the driving force accountable for variant bovine spongiform encephalopathy. This has lead to a global effort to diminish the use of BSA in pharmaceuticals. As a protein exceedingly preserved in evolution, BSA and human serum albumin (HSA) share 80% progression homology. Their molecular weights vary by less than 1%. BSA has served as a replica protein for studying the immune response to proteins. Catalase like haemoglobin both contains heme group. Hemes are a diverse group of teterapyrrole pigments, being present as the prosthetic group of both the globins (haemoglobin and myoglobin), and the cyctochromes (including those involved in respiratory and photosynthetic electron transport. In all the hemeproteins, the role of the heme is to either attach or release a ligand to its central iron atom, or to undergo oxidation-reduction reaction. Catalase is found in peroxisomes where they degraded fatty acids and amino acids. The by-products of these reactions hydrogen peroxide, which is toxic to the cell. The presence of the high amount of the enzyme (catalase) in the peroxisome rapidly converts the toxic hydrogen peroxide into harmless water and oxygen. Haemoglobin is a globular protein tetramer consisting of two alpha (α) and two beta (β) chain subunits which gives it speciality function in binding and transporting oxygen (o2) from the lungs to the respiring tissues and in a reverse way of transporting CO2 from the tissues. As all proteins absorb at 280nm, spectrophotometer technique use here is therefore, ideal to determine their peak absorbance, as per Beer-Lambert law.

Experimental method: we used the following materials: Suspension of Carborxymethyl Cellulose CM23 in 0.01M acetate buffer, pH5.0. A mixture of Proteins, containing Bovine Serum albumin, Catalase and Haemoglobin. Buffers: 0.01M acetate buffer, pH 5.0, 0.01M acetate buffer, pH 6.0 and 0.10M Tris buffer, pH 8.0.

Data

Bovine Serum Albumin pI 4.7 Catalase pI 5.4 Haemoglobin pI 6.7

Method:

We follow the protocol in experiment 2.4 workbook P34 as follows: Packing the column We inserted a glass fibre disc in to the barrel of a 5mL (we used a syringe as the column, using a glass rod push down the barrel until it is lied flat on the base. We mounted the syringe on the clamp stand and making sure the syringe outlet was close with the clip. We inverted the suspension of CM-cellulose tenderly a number of times, to resuspended and quickly poured some in to the syringe barrel, filled it about half way. We place a beaker under the column, opened the clip and allowed the buffer to flow through as fast as possible, without having to worry about the column running dried. We allowed the buffer to drain through until none remains above the surface of the packed cellulose. The packed column volume should be about 2-3ml. We close the clip and inserted a second glass fibre disc into the syringe, pushing it down gently to rest on the top of the packed cellulose. This helps prevent the surface been disturbed.

Washing of the columns: A pasture pipette was use to tenderly filled up the syringe barrel with 0.01M acetate buffer, pH5.0. Opened the clip and allowed the protein mixture to drain into the column, the close the clip slowly and added 0.5ml of 0.01M acetate buffer, pH 5.0 and allow this buffer to drain through by opening the clip. The glass fibre disc now appeared white.

Loading the column: Very slowly applied 0.3ml of the protein mixture to the top of the glass fibre disc, open the clip and allow the protein mixture to drain through the column. Closed the clip, slowly and added 0.5ml 0.0lM acetate buffer, pH5.0, then open the clip and allow this buffer to drain through the column.

Eluting the columns: We followed the protocol on experiment 2.5, on workbook Bioc 1302 as follows: A test tube rack was fill with 20 small plastic tubes, and numbered from 1-20 using Gilson pipette. A 2.0ml was measured into one tube, marked the level of the fluid with a marker pen. This tube was used as a template to mark the rest to show level required for collecting 2ml samples. We set spectrophotometer to 280 nm and zero with 0.01M standard acetate buffer, pH5.0 and began collection the fractions. We kept the flow rate to around 2ml/min. Set spectrophotometer to 280 nm and zero with 2ml of 0.01 acetate buffer, pH 5.0, and used the UV transparent plastic cuvettes provided. We added 0.3ml of protein mixture to the top of the column, and allowed this to enter the gel. Immediately filled the syringe with 0.01M acetate buffer, pH 5.0 and began collecting 2ml fractions. We kept the flow rate to around 2ml. We continued washing the column with this buffer (0.01M acetate buffer, pH 5.0) until the A280 is just about zero. We zero the spectrophotometer with 0.01M acetate buffer, pH 6.0 at A280. To elute Catalase, we filled the column up with 0.10M acetate buffer, pH 6.0. Continued collecting 2ml fractions measured the A280 of each. The A280 should again return towards zero. To elute haemoglobin, we filled up the column with 2ml tris buffer, pH8.0, and zero again the spectrophotometer with tris buffer, pH 6.0 at A280. We continued collecting fractions of 2ml until the A280 near zero again. We retained all fractions until the experiment is completed, and record A280 values for each. Fractions which gave, the highest A280 values from each peak (i.e. the peak obtained with buffer different concentrations of pHs). These were kept for the SDS-PAGE experiment.

Detection of Catalase activity: We pipette 50µl of 6% hydrogen peroxide solution into wells in the 96-well plats provided and added 20µl of each fraction to be tested to one of the wells. We kept an accurate record of which fraction was been tested in each well. The Fractions containing Catalase immediately froths as oxygen is released from the hydrogen peroxide solution.

Identifying proteins: We calibrated the spectrophotometer to 405 nm with standard haemoglobin solution, absorbance taken of haemoglobin peak fraction collected from the eluted sample. Absorbance at this region indicates the presence of haemoglobin.

Separating proteins molecules using SDS-PAGE: We follow the protocol in experiment 2.6 workbook Bioc1302 as follows: The apparatus used was a Biorad mini-protean ll system, containing two 10% SDS-polyacrylamide gel run in a buffer of tris-glucine, pH 8.3, containing 0.1% SDS.

To prepare samples for SDS-PAGE Data: 50µl sample, 50µl loading buffer and 10 µl tracker dye mixed well in an appendorf and label which wells contained our samples. We applied 10µl samples to the gel, 10µl samples of molecular weight markers and original BSA, Catalase and Haemoglobin solutions (provided) were also loaded onto the gel. This gel was run for 30 minutes at 200V, removed and stained with Coomassie blue, destained it and finally presented for measuring the distance migrated by each protein band. Proteins are identified base on their molecular masses. Each band on the gel of the SDS-PAGE correspond to the molecular weight of the protein and this was determined by taking the log10 of the molecular mass against the distance travelled by each protein in the gel band.

RESULTS:

Calculations: work example: 100ml of 0.01M acetate buffer, pH6.0. BSA pI = 4.76. pH = pKa + log[conjugate base]/[conjugate acid] 6.0 = 4.76 + log[A-]/[H+] A-/H+ = 17.38/1, A- = (17.38/18.38) X 100ml = A-= 94.56ml H+= (1/18.38) X 100ml = H+ = 5. 44ml

Ratio of Acid: Base = 5.44ml: 94.56ml

A Table showing fractions of different proteins, with different buffer solutions, at varied absorbance.

Fractions

1

2

3

4

5

6

7

8

9

10

Absorbance

1.610

0.574

0.058

0.013

0.003

-0.037

-0.026

0.007

1.425

0.861

Fractions

11

12

13

14

15

16

17

18

19

20

Absorbance

0.158

0.168

0.076

0.050

0.856

0.988

0.165

0.105

0.123

0.076

From fractions 1-5, acetate buffer pH 5.0 was used. From fraction 6-14, acetate buffer pH6.0 was used, and from fraction 15-20 tris buffer pH8.0 was used.

Peaks at A280: We found three peaks of absorbance within the wavelength of 280nm tested. BSA has a peak at 1.610; Catalase has a peak at 1.426 and Haemoglobin at 0.988. All the peaks were symmetrical and linear.

Observation:

Step1: After adding 0.5ml of 0.01M acetate buffer of pH 5.0, a dark brown colour band appeared to slightly enter the CM column. The first clear colour solution was eluted, which indicates BSA protein, as pH5.0 of acetate buffer used here is above the pI of BSA, and therefore, its net charge d was turned negative and unbounded from the column first.

Step2: After adding 0.5ml of 0.01M acetate buffer pH6.0, a light brown colour solution was eluted. This indicates Catalase protein; as the pH6.0 of acetate buffer used here, it is above the pI of catalase, and therefore, its net charge was turn negative. This net negative charged on catalase made it unbounded from the column and eluted second.

Step3: After adding 0.5ml of 0.10M Tris buffer pH8.0, a dark brown colour solution was obtained. This indicates the haemoglobin protein, as the pH8.0 of Tris buffer is above the pI of haemoglobin and therefore, its net charge negative and unbounded from the column and eluted.

Detection of Catalase activity

Catalase activity was detected in fractions: 9, 10, 11, 12 and 13 of eluted Catalase fractions.

SDS-PAGE IMAGE 7

A table showing molecular masses of standard proteins (log10) with distance migrated (mm) from the gel.

Molecular masses (KDa)

Distance migrated (mm)

Relative mobility

Log10Mr

250

22

0.20

2.40

150

33

0.30

2.18

100

42

0.38

2.00

75

51

0.46

1.88

50

63

0.57

1.70

37

73

0.66

1.57

25

88

0.80

1.40

20

95

0.86

1.30

15

107

0.97

1.18

10

110

1.00

1.00

Sample calculations: log10 (250) = 2.397940009 ~ 2.40

Discussion:

There were three peaks observed in the absorbance of the spectrophotometer with different concentration of buffer solutions, which indicates the presence of all the three proteins of our interest. A catalase detection test performed on the eluted fractions shown catalase activity only on the catalase fractions: 9,10,11,12 and 13. This was due to the faintly brown protein which catalyses the decomposition of hydrogen peroxide releasing bubbles of oxygen. BSA is a colourless solution protein and therefore, does not react with hydrogen peroxide treatment. Haemoglobin has a reddish brown protein and does not react with hydrogen peroxide treatment either. The spectrophotometer used was an ideal technique, as absorbance could be calculated using Beer-Lambert law; A=εcl. The peak fractions used for the SDS-PAGE gel experiment for detection of the proteins molecular masses were all contaminated. Although the thick bands of proteins were seen on the gel, but the fact that our proteins were not 100% pure, could lead us to wrong molecular weights analysis. This could be due to certain factors affecting the purity of our proteins. In the spectrophotometer analyses, slight errors in calibration could have an impact on the result obtained, even though this could be insignificant. As the volumes measured were very small, pipetting error could have also impacted on the result in general. There could be scratches or dirt on the transparent cuvettes which could also affect the absorbance readings and the peak fractions might not have contained enough protein as it should have. Alternatively, the gel may have not been polymerised fully or may possibly unevenly polymerise. This is frequently due to the use of old ammonium persulphate. This could also be due to the intensity of high oxygen dissolved in the gel, or by the use of low pH buffers that slow down the polymerisation reaction. On the other hand, the gel may polymerise too quickly, either due to too much ammonium persulphate or TEMED, or high laboratory temperature (the gel mixture can be cooled before adding catalysts). The buffer solutions prepared might be in higher temperature to the temperature of the protein mixtures and the resin column, which might the elution of the impure samples.

Future modification to improve the accuracy and precision: Automatics pipettes and plate readers could minimize the level of experimental error in this experiment. A fresh solution of ammonium persulphate should be prepared daily to avoid the polymerisation problems. Solutions can be degassed before use if necessary, and gel solution should be gently poured down inside the side of the apparatus to avoid introducing air. Buffer solutions should be prepared and left under the same room temperature as the proteins for some to be able to get the same unique temperature.

Conclusion:

Protein bands of our BSA, Catalase and haemoglobin samples were shown on the gel page, even though there were contaminants. We were able to separate our proteins, but they were not 100% pure. A further purification research is required in order to obtain 100% pure protein samples and effective methods are needed to get rid of the contaminants. We have achieved our aims in separating our protein samples, but not to the extent of purity.

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