Analysis Of Egg White Proteins: Gel Electrophoresis, Biuret Assay And Lysozyme Activity Assay

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.