Practical Exercise 3: Analysis of Egg White Proteins - Lab Report

This laboratory report details a practical exercise analyzing egg white proteins, specifically ovalbumin and lysozyme. The study aimed to quantify these proteins and assess their biochemical properties through gel electrophoresis (SDS-PAGE), biuret assays, and lysozyme activity assays. The report...Read More

Running head: Practical Report
Practical Exercise 3
Analysis of Egg White Proteins – Part II:
Quantification, Purity and Size Determination and
Activity Assays
Name of the student:
Name of the university:
Author note:

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LABORATORY REPORT
Table of contents
Table of figures..........................................................................................................................2
Introduction................................................................................................................................3
Aims of the study.......................................................................................................................3
Materials and Methods..............................................................................................................4
Results........................................................................................................................................4
Analysis of ovalbumin purity and size by gel electrophoresis.....................................................4
Discussion..................................................................................................................................9
Analysis of ovalbumin purity and size.........................................................................................9
References................................................................................................................................12

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LABORATORY REPORT
Table of figures
Figure 1. Showing SDS PAGE gel electrophoresis. Lane 1-Mraker, lane 2- egg white, lane
3 withouth lysozome, lane 4 lysozome separted, lane 5 egg white afetr removign lysozome,
lane 6 ovomucin........................................................................................................................4
Figure 2 Graph display of arbsorbance and BSA......................................................................5
Figure 3 Table showing protein concentration and arbsobance................................................6
Figure 4 Enzyme activty of lysozyme assay..............................................................................6
Figure 5 Enzyme activiy assay of lysozyme..............................................................................7
Figure 6 Lysozyme fraction calculation....................................................................................8

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LABORATORY REPORT
Introduction
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.
Aims of the study
The following were the aims of this study;
i) To quantify lysozyme and ovalbumin proteins.
ii) To assess biochemical properties of ovalbumin and lysozyme through
electrophoresis, burette assay and lysozyme activity assay.

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LABORATORY REPORT
Materials and Methods
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.
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.
Results
Analysis of ovalbumin purity and size by gel electrophoresis

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LABORATORY REPORT
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 1. Showing SDS PAGE gel electrophoresis. Lane 1-Mraker, lane 2- egg white, lane 3
withouth lysozome, lane 4 lysozome separted, lane 5 egg white afetr removign lysozome, lane 6
ovomucin
2 76543 81

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LABORATORY REPORT
1 2 3 4 5 6
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
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 0.1 0.1 0.1 0.1 0.1 0.1
Protein concentration of BSAmg
A540 absorbance units (AU)

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LABORATORY REPORT
original sample
(mg/mL): above
amount X dil.
Factor
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

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LABORATORY REPORT
0 30 60 90 120 150 180 240 270 300
0
0.5
1
1.5
2
2.5
Lysozyme fraction D
Lysozyme fraction C
Lysozyme fraction B
Lysozyme fraction A
Time in seconds
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

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LABORATORY REPORT
Discussion
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 =
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%

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LABORATORY REPORT
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).
Determination of lysozyme activity
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

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LABORATORY REPORT
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.

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LABORATORY REPORT
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.