Name: ANDAL, Daniel Seth Date Performed: Feb.

10, 2020
Group number: 2 Date Finished: Feb. 14, 2020
Exercise 4.1
TECHNIQUES FOR PROTEIN ANALYSIS: ISOLATION OF PROTEINS

I. RESULTS
Table 4.1.1. Preparation of crude egg albumin by ammonium sulfate precipitation.

Steps
1. Break up two medium sized eggs and
separate egg whites carefully from the yolk.
2. Stir the egg white to break the membrane.
3. Filter through cheesecloth then measure 40
mL of filtrate (filtered undiluted egg white).
4. Add 4 mL of 1M acetic acid slowly with
constant stirring
5. Remove the precipitate using cheesecloth.
6. Obtain 30 mL of filtrate and bring the
solution to 0% to 40% saturation by adding
7.26 g of (NH4)2SO4 in small portions with
constant stirring under an ice water bath.
7. Filter through cheesecloth.
8. Bring the solution to 40% to 60% saturation
by adding 3.90 g of (NH4)2SO4 in small portions
with constant stirring under an ice water bath.
9. Allow the solution to stand with occasional
stirring for 30minutes under an ice water bath.
10. Filter using pre-weighed filter paper.
11. Air dry and weigh the precipitate.
Mass of filter paper: 1.10 g
Mass of filter paper + crude egg albumin: 15.82 g
Mass of crude egg albumin (actual yield): 14.72 g
Table 4.1.2. Isoelectric precipitation of casein.
Observation
Clear (yellowish in color)
- Contaminated with egg yolk
Turbid, less viscous, appearance of bubbles
Bubbles disappeared
Turbid, appearance of white precipitate
White precipitate as residue and clear solution
as filtrate
(NH4)2SO4 instantly dissolves when added to
the filtrate. Eventually forming a milky white
solution. Dissolution occur longer and few
precipitate were present at the bottom.
White precipitate as residue and milky white
solution as filtrate
(NH4)2SO4 instantly dissolves when added to
the filtrate. Eventually forming a whiter
solution and became less viscous
The solution becomes less viscous and
maintains its coloration
The filter paper appeared light yellow as it is
still wet after filtration
White crystals along with remains of creamy
residue on the filter paper

Test tube no. pH Observation

1 2.7 Clear solution
2 4.7 Turbid solution
3 6.7 Clear solution
II. SAMPLE CALCULATIONS
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑐𝑟𝑢𝑑𝑒 𝑒𝑔𝑔 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (𝑔)
%(w/v) = 𝑥 100
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑓𝑖𝑙𝑡𝑒𝑟𝑒𝑑 𝑢𝑛𝑑𝑖𝑙𝑢𝑡𝑒𝑑 𝑒𝑔𝑔 𝑤ℎ𝑖𝑡𝑒 (𝑚𝐿)

14.72 𝑔
= 30 𝑚𝐿
𝑥 100

%(w/v) = 49.07 %

III. DISCUSSION

Proteins are isolated by means of precipitating it from a mixture of soluble substances. A

common way for precipitating proteins is the salting-out process. This method involves the use
of ammonium sulfate, an inorganic salt, to saturate the protein solution. During the process of
salting-out, Wingfield (2001) salt increases the surface tension of water which leads to increased
hydrophobic interaction between protein and water. As a response, the protein decreases its
surface area to minimize contact with the solvent where it associates with itself and eventually
forming a precipitate. Moreover, increasing concentration of ammonium sulfate decreases the
solubility of proteins due to these hydrophobic interactions forcing the proteins for higher rates
of self-association.
About 10% of the egg white is made up of proteins where 54% of this fraction is
ovalbumin. As to the other proteins present, they can be named as ovotransferrin, ovomucoid,
ovomucin, lysozyme, and globulin. Ovotransferrin provides antimicrobial defense for the egg’s
developing embryo suiting its ability to isolate the iron required for microbial growth, thus
depriving its infestation to the embryo as explained by Wu and Acero-Lopez (2012). On the other
hand, Caubet and Wang (2011) expound that ovumucoid is stable against heat and digestion
against proteinases, and as a dominant allergen due to its strong disulfide bonds stabilizing a
highly glycosylated ovomucoid. Sharif, Saleem, and Javed (2018) descrived ovomucin as another
glycosylated protein that has a fibrous conformation which provides antimicrobial growth as well
due to its viscous nature and specific binding to tumor and cholesterol activities. Its fibrous
conformation regards it to be insoluble proteins that are mechanically strong a-nd provides
structural and protective support for the embryo. Additionally, lysozymes also have antimicrobial
properties where Silvetti et al. (2017) explain its hydrolyzing effect to the glycosidic bonds of
peptidoglycan in Gram-positive bacteria. Lastly, globulin, as globular proteins, provide transport
throughout the membrane of the egg as it has water-soluble properties.
The abundant protein portion of the egg white is the ovalbumin. In order to isolate
ovalbumin from egg whites, a series of steps and addition of reagents must be followed. The first
step after obtaining the egg white is mild acidification using acetic acid in order to disrupt the cell
membrane to allow the extraction of the crude protein which after is filtered through a
cheesecloth. Upon obtaining the filtrate, gradual addition of ammonium sulfate with constant
stirring was reinforced while the sample was under an ice water bath in order to prevent
denaturation to occur. The ammonium sulfate addition was necessary in order to isolate the
protein by salting out where stronger hydrophobic interaction between the protein and water
occur as the salt dissolves in the water and the protein becomes the precipitate. As the gradual
addition of salt, it is essential to avoid the accumulation of high salt concentration. Furthermore,
two percent range of saturation was used during this addition of ammonium sulfate. Firstly, 0-
40% addition accounts for the increase of the protein’s solubility due to additional ions from the
salt shields the multiple charges of amino acid residue leading to decreased self-interaction of the
amino acids preventing aggregation and precipitation while the 40-60% addition’s purpose is for
the sample to have a decreased solubility for the protein to precipitate, hence, the difference on
observation between these saturations on Table 4.1.1.
The weight of the crude sample after 5 days of allowing to stand is 14.72 g with a 49.07%
(w/v) to the overall protein composition of the egg white. Literature value states that 54% of the
egg white is ovalbumin. However, the obtained %(w/v) lacks 4.93% to conform to this. This yield
could have been the result of protein denaturation during the process. It could specifically
accounted on the separation of the egg yolk and the egg white as recorded on Table 4.1.
Furthermore, the room temperature and possible contamination by hand and by glassware
sanitation are also possible sources why the yield was less than the literature value.
Solubility of proteins is affected by the pH of the solution it is subjected upon where
proteins are least soluble at the point where the pH is equal to its isoelectric point. In this
experiment, casein was treated with NaOH for it to dissolve and subjected to three buffer solutions
of different pH. Theoretically, the ipH of casein is established to be around the region of pH 4.6
as stated by O’Kennedy (2011). Due to this information, casein is expected to undergo
precipitation when subjected at the test tube containing acetate buffer with pH 4.7. Based on the
data obtained and recorded at Table 4.1.2, it was observed that the solution was turbid and
resulted to precipitation on the test tube with acetate buffer unlike the other test tubes which
have a clear solution. This conforms to the literature value where protein precipitates at its
isoelectric point with the basis of the mixture’s turbidity and the presence of precipitate.

IV. REFERENCES/LITERATURE CITED

CAUBET, J. C., WANG, J. 2011. Current understanding of egg allergy. Pediatric clinics of North
America, 58(2), 427–xi. doi:10.1016/j.pcl.2011.02.014.
O’KENNEDY, B. T. 2011. Caseins. Handbook of Food Proteins. Cambridge, UK; Philadelphia:
Woodhead Publishing, 2011.
SHARIF, M. K., SALEEM, M., JAVED, K. 2019. Chapter 15 - Food Materials Science in Egg Powder
Industry. Role of Materials Science in Food Bioengineering. Handbook of Food
Bioengineering, 2018, Pages 505-537. https://doi.org/10.1016/B978-0-12-811448-
3.00015-2.
SILVETTI, T., MORANDI, S., HINTERSTEINER, M., BRASCA, M. 2017. Chapter 22 – Use of Hen
Egg White Lysozyme in the Food Industry. Egg Innovations and Strategies for
Improvements, 2017, Pages 233-242. https://doi.org/10.1016/B978-0-12-800879-
9.00022-6.
WINGFIELD, P. 2001. Protein precipitation using ammonium sulfate. Current protocols in protein
science, Appendix 3, Appendix–3F. doi:10.1002/0471140864.psa03fs13.
WU, J., ACERO-LOPEZ, A. 2012. Ovotransferrin: Structure, bioactivities, and preparation. Food
Research International. Volume 46, Issue 2, May 2012, Pages 480-487.
https://doi.org/10.1016/j.foodres.2011.07.012.