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Size determination of proteins by SDS-PAGE


Analysis of Egg White Proteins

To quantify two main egg white proteins, ovalbumin and lysozyme, and assess their selected biochemical properties by key analytical techniques of gel electrophoresis, Biuret assay and lysozyme activity assay.

Size determination of proteins by SDS-PAGE: Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) is commonly used to separate proteins according to their electrophoretic mobility, which is a function of length of the polypeptide chain, or molecular weight. Treatment of proteins with SDS and a reducing agent relaxes their secondary structures, giving a uniform positive charge and reduces disulphide bridges. This leads to all proteins having identical charge to mass ratios, which results in separation by size alone on the gel when an electric potential is applied.

Quantitation of total protein content in a sample by Biuret assay: The Biuret test is used for detecting the presence of peptide bonds. In a positive test, a copper (II) ion is reduced to copper (I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. A violet colour indicates the presence of proteins. The Biuret test is commonly used to determine the concentration of protein in mg/mL in a sample. The intensity of the colour, and hence absorbance at 540 nm is directly proportional to the protein concentration, according to the Beer-Lambert’s law. The range of sensitivity of the test is 1 – 10 mg total protein, so samples need to be diluted if they have a lot of protein.

Assay of the enzymatic activity of lysozyme: As mentioned in Practical 2, lysozyme is an important protein produced in many animals and is abundant in secretions such as tears, saliva, mucous and in egg whites. It is considered to be a protein for defence against bacteria, due to its ability to lyse bacterial cells. It is an enzyme which damages bacterial cell walls by catalysing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan (the main component of bacterial cell walls), and between N-acetyl-D-glucosamine residues in chitodextrins (fragments of chitin: the main component of fungal cell walls).

Lysozyme activity is typically measured by its action on the bacterium Micrococcus lysodeikticus. The structural polysaccharide of the cell wall of this microorganism is very susceptible to lysozyme- catalysed hydrolysis. This is the basis of the assay is that, a suspension of the bacteria will gradually look clear if treated with lysozyme, as the cells are broken up and cell contents are released. The lysozyme activity (rate of cell lysis) is measured by observing the decrease in absorbance at 450nm of a bacterial culture treated with lysozyme solution. Faster the decrease in absorbance, greater the activity present.

In this practical, you will use the samples purified in Practical 2 for the following 3 exercises:

  1. Determining the purity and approximate size of the egg white ovalbumin prepared in Prac 2, compared to the whole egg white proteins, by SDS-PAGE.
  2. Quantitating the protein content of the (partially) purified ovalbumin and lysozyme samples from Prac 2, as well as the whole egg white, by the Biuret test. This test will also help assess how much protein is being lost in each purification step of lysozyme and give an idea of the purity of the sample, once the lysozyme activity in each sample is tested.
  3. Testing the enzymatic activity of the four fractions (A, B, C, D) of lysozyme prepared in Prac 2, on a bacterial cell culture as substrate.

Materials:

  • Slant of Micrococcus lysodeikticus
  • Lysozyme fractions A-D from Prac 2
  • 0.066 M sodium phosphate buffer (pH 6.24)

Methods

    1. Add 3 mL of the phosphate buffer to the slope of the M. lysodeikticus slant and swirl it round to remove the bacteria from the slant surface.
    2. Pour the suspension into a clean test-tube, mixing carefully so that a uniform suspension is obtained.
    3. Take 50 to 100 µL of this suspension and make various dilutions with water in finala volume of 2mL, in glass spectrophotometer tubes (cuvettes). Use a spectrophotometer and note the absorbance of each of these dilutions at 450nm. Use water as ablank.
    4. Work out from the above step which dilution gives you A450 of about 0.5 units. Then prepare 15 mL of bacterial suspension at this dilution in a 15 mL centrifuge tube. This is to be used as substrate for the lysozyme activity assays. This is an important step, so consult your demonstrator regarding your readings and dilutions.
    5. Find your lysozyme fractions A-D from Prac 2. Measure and note their volumes.You need this information for your report.
    6. Add 0.5 mL of the fraction A solution to 2 mL of the substrate (bacterial suspension above) in a glass spectrophotometer tube. Mix well, note down the time (time zero), and immediately start recording the A450 every 30 seconds from time zero, for the next 5 minutes. Write the data in two copies of Table 2 (sheet given in the lab).  It is essential that you have your own copy and can produce it if needed. Note: it may be necessary to dilute the lysozyme Fraction A at this point if the activity is very high. If so, Note the dilution factor.
    7. Repeat the above step (Step 6) for fractions B, C and D. Record the absorbance values as above, in the same table. Dilute these fractions too if required, and then record the absorbance readings.
    8. Plot the graphs of Absorbance versus Time, for all four samples on one graph (using different colours).
    9. Please discard all bacterial suspensions (used/unused) in the appropriately labelled container.
Size determination of proteins by SDS-PAGE

Ovalbumin and lysozyme contribute significant proportion of the major proteins in the egg, they account for 54% and 3.4% of total egg protein. Theoretically ovulbumin has a molecular weight of 45kDa and is easily isolated from the egg white. More than half are hydrophobic while one third are charged, (Abeyrathne, Lee & Ahn, 2014). Ovalbumin has been widely been used in an array of assay as it has standard properties and has significant effect on immunological functions and nutritional effects. Lysozome entail single polypeptide having 129 amino acids and has a molecular weight of 14.4kDa. it a basic protein containing an iso electric point of 10.7 with four disulfide bridges thus having high stability, (Datta et al., 2009).

Quantification of these two proteins has been undertaken. Further size determination using sodium Dodecyl Sulphate Polyacrylamide  Gel  Electrophoresis  as referred to as (SDS-PAGE) is a common way of separating proteins based on the electrophoresis movement. These two proteins will be determined using this technique. Further burette assay will be initiated in order to allow detection of peptide bonds using copper ii ions. It is essential for determination of protein concentration, (Wu & Acer-Lopez, 2012).  Lysozome enzymatic activity is crucial in enabling ability of lyse bacterial cells, it changes bacteria walls through catalysis and hydrolysis of 1,4 beta linkages when compared with N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan, (Cegielska-Radziejewska, Lesnierowski  and Kijowski, J., 2008). Thus the lysozyme activity reflects the action of bacterium micrococcus lysodeikticus.

The following were the aims of this study;

  1. To quantify lysozyme and ovalbumin proteins.
  2. To assess biochemical properties of ovalbumin and lysozyme through electrophoresis, burette assay and lysozyme activity assay.

Gel electrophoresis was used to analyze ovulabumin size and purity. Ovulbumin protein was mixed with reducing buffer SDS PAGE buffer using 0.1% at pH 8.5. Further molecular weight marker was used to measure molecular weight and gel stain. SDG PAGE was measured at 10 µL and added to ovalbumin protein. Micro centrifuge was undertaken in boiling water for 30 minutes. Further the gel was run at approximately 30-40 minutes using 200 volts and the gel image was captured using image scanner.

Biurett assay was undertaken in order to assess concentration of protein based on the ovulbumin and lysozome. Standard bovine serum albumin measuring 10mg/Ml was used  with burette agent. BVA was mixed with the distilled water to get 1 ml solution altering the concentration of distilled water.  Five test tubes were labeled and used , with addition of 3 mls of fresh burette reagents, with absorbance rates being recorded.

Quantitation of total protein content in a sample by Biuret assay

Lysozome enzymatic activity was determined using bacterium slant of Micrococcus lysodeikticus, lysozyme fractions from biurett solutions and 0.066 molar of sodium phopshate buffer at pH 6.24. 3 mls of the phosphate buffer was added to the bacteria and mixed , then poured into test tubes. Various dilutions were made between 50-100 µL and mixed with water of 2mL. Glass spectrophotometer was done with each absorbance being noted at 450nm. 15 ml of the bacteria solution was used as a substrate for the lysozyme activity assays. Using the lysozyme fraction from Biurett assay, addition of 5mL of bacterial substrate was added and mixed in glass spectrophotometer and absorbance values undertaken.

Analysis of ovalbumin purity and size by gel electrophoresis

Determination of molecular weight of ovalbumin was done through the separation of the gel using set molecular standards. The gel was processed and then distained, with the relative migration distance being determined. SDS PAGE offers patterns which are characterized by one of more proteins bands. Bands through electrophoresis highly characterize the sample, while strands with faint pattern often may not represent clear pattern.

The gel observation reflects large patterns which tend to degrade. Cleavage of large proteins results to smaller bands. Polypeptides bonds with one or more non covalent associations can occur.

Biuret assay analysis on protein concentration

b) Standard curve for BSA concentration against respective BSA amounts

Figure 2 Graph display of arbsorbance and BSA

Gradient calculations for the curve

Gradient line= changes in x/changes in y

= (5.5-3.0) / (0.7-0.2)

=  3mg/mL

c) Estimation of protein concentration through extrapolation of absorbance values

Tube No.

7

8

9

10

11

12

Protein sample

Whole egg

Ovalbumin

Lysozome Fraction A

Lysozome Fraction B

Lysozome Fraction C

Lysozome Fraction D

A450

Protein content

(mg/mL): estimated

from standard curve

0.9

0.9

0.9

0.9

0.9

0.9

Protein content in

original sample

(mg/mL): above

amount X dil. Factor

0.1

0.1

0.1

0.1

0.1

0.1

Total volume of

sample (mL)

1

1

1

1

1

1

Total protein

content (mg)

2

2

2

2

2

1

Figure 3 Table showing protein concentration and arbsobance

d) Enzymatic activity assay of lysozyme in purification fractions

Absorbance of Micrococcus lysodeikticus culture at 450 nm

Time

0

30

60

90

120

150

180

240

270

300

Lysozyme fraction A

0.550

0.443

0.430

0.441

0.434

0.428

0.420

0.420

0.421

0.422

Lysozyme fraction B

0.444

0.446

0.449

0.450

0.451

0.450

0.450

0.450

0.450

0.450

Lysozyme fraction C

0.402

0.423

0.423

0.423

0.423

0.423

0.423

0.425

0.925

0.426

Lysozyme fraction D

0.492

0.478

0.447

0.406

0.350

0.305

0.260

0.219

0.184

0.131

Figure 4 Enzyme activty of lysozyme assay

Graphical presentation

 

Figure 5 Enzyme activiy assay of lysozyme

Calculation presentations

Time

Gradient of tangent at t=o

Activity (U) in the 0.5mL in the tube

Activity (U/mL)

Protein content (mg/mL) from biuret Test

Specific activity (U/mg))

Volume of sample

Total activity isolated (U)

Lysozyme fraction A

-1.37AU/min

1370U

2740U/mL

3mg/mL

913U/mg

1 mL

2740

Lysozyme fraction B

-1.30AU/min

1300U

2600U/mL

3mg/mL

866U/mg

1 mL

2600

Lysozyme fraction C

-1.32AU/min

1320U

2640U/mL

3mg/mL

880U/mg

1 mL

2640

Lysozyme fraction D

-1.4AU/min

1400U

2800U/mL

3mg/mL

933U/mg

1 mL

2800

Figure 6 Lysozyme fraction calculation

Analysis of ovalbumin purity and size

Based on the gel electrophoresis, ovualbumin ran was observed forming long strands of different density. Ovalbumin contain is iso electric point of 4. In the gel display strands of ovualbumin was observed running with different strands.

There was existence of other proteins which were co-purified with ovalbumin in this experiment. This was observed with the various strands observed in the gel electrophoresis display marker, (Roy, Rao & Gupa, 2003).

Ovalbumin purity is very crucial in assessing analytical standards of whole protein in the egg. For this case the % purity will be calculated using =

Assay of the enzymatic activity of lysozyme

Percentage purity = mass of useful product/total sample mass  X 100

= Protein content of original sample=1

= protein content estimated from curve= 0.9

= 0.9/1X100

= 90%

Ovalbumin purity could be enhanced through performance of gel infiltration after Ni-NTA. In this way size exclusion chromatography is separated from the protein. The protein sample can be applied on top through the porous beads which are insoluble and hydrate polymer. Further, ion-exchange chromatography, affinity chromatography and dialysis of the proteins, (Johnson & Larson, 2005).

The activity of lysozyme is initiated through ion exchange conditions. Carboxy-methycellulose is essential for maintaining the negative ions in the solution. In the results observed the fraction with most lysozyme activity was fraction D. This is due to the binding of net positive charge, which enhances the biding to carboxy-methycellulose, (Hall, Jones & Forest, 2015).

The purification of lysozyme was not effective in that the ions exchange activity with carboxy cellulose was effective. More positive ions were released thus ensuring the pH solution binding to the compound. With the instability of lysozome in the Ph solution, lysozyme was isolated effectively. The observed low yield of lysozyme was characterized by the loss of lysozyme due to binding effect of trapping during the purification process, (Yu, Wang & Ulrich, 2014).

Fraction B and C showed reactivity through measurement of the optical density of the spectrophoresis. There was an elevated rate of reaction observed from the experiment. The presence of lysozyme bacteria present tin the process, offers specificity to binding thus allowing for enzymatic activity while the density is being decreased. Thus the presence of all the activities observed reflects the binding effect of lysozyme which binds the cellular walls of the protein.

Fraction A and D shows increased specificity which shows the percentage of purification undertaken during the processes. With fraction range of 1500-30,000 da using chromatography and molecular weight shows that egg fraction entailing A and D have purified lysozyme showing high specificity. The decline in specificity is observed after a while due to the presence of impurities in the trappings thus lowering the activity rate of lysozyme, (Mol, Verssimo, Eller , Minim & Minim, 2017).

Error source in this experiment was minimized to manageable levels. The activity of lysozyme was undertaken using the assessment of optical density. Sources of error could have experienced were the pipe ting of the minute amounts in pipette which could be challenging to achieve. The cellular molecular process of cell biology on lysozyme is critical in the field of study. The micrococcus bacteria used is crucial in establishing peak enzymatic activity. The exact nature of proteins however remain inconclusive, there is need for in depth understanding of protein assay.

Thus this lab experiment demonstrated how protein exclusion can be undertaken and purified based on this molecular mass. Further functionality of specific substrate activity was undertaken to determine the purified fraction entailed in enzyme activity.

References

Abeyrathne, E.D.N.S., Lee, H.Y. and Ahn, D.U., 2014. Sequential separation of lysozyme, ovomucin, ovotransferrin, and ovalbumin from egg white. Poultry science, 93(4), pp.1001-1009.

Cegielska-Radziejewska, R., Lesnierowski, G. and Kijowski, J., 2008. Properties and application of egg white lysozyme and its modified preparations-a review. Polish Journal of Food and Nutrition Sciences, 58(1).

Datta, D., Bhattacharjee, S., Nath, A., Das, R., Bhattacharjee, C. and Datta, S., 2009. Separation of ovalbumin from chicken egg white using two-stage ultrafiltration technique. Separation and Purification Technology, 66(2), pp.353-361.

 Hall, B., Jones, L. and Forrest, J.A., 2015. Kinetics of competitive adsorption between lysozyme and lactoferrin on silicone hydrogel contact lenses and the effect on lysozyme activity. Current eye research, 40(6), pp.622-631.

Johnson EA, Larson AE 200. Lysozyme, in: Davidson PM, Sofos JN, Branen AL, eds, Antimicrobials in Food, third edition. New York, Taylor & Francis Group,pp. 361–387.

Mól, P.C.G., Veríssimo, L.A.A., Eller, M.R., Minim, V.P.R. and Minim, L.A., 2017. Development of an affinity cryogel for one step purification of lysozyme from chicken egg white. Journal of Chromatography B, 1044, pp.17-23.

Roy, I., Rao, M.V.S. and Gupta, M.N., 2003. An integrated process for purification of lysozyme, ovalbumin, and ovomucoid from hen egg white. Applied biochemistry and biotechnology, 111(1), pp.55-63.

Wang, J. and Wu, J., 2012. Effect of operating conditions on the extraction of ovomucin. Process biochemistry, 47(1), pp.94-98.

Yu, X., Wang, J. and Ulrich, J., 2014. Purification of Lysozyme from Protein Mixtures by Solvent?Freeze?Out Technology. Chemical Engineering & Technology, 37(8), pp.1353-1357.

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