Jimenez, Marc R.

September 19, 2016

CHEM 160.1 – 2L (Group 3)

Exercise 4.1
ISOLATION OF PROTEINS
Table 4.1.1. Preparation of crude egg albumin by ammonium sulfate precipitation.
Steps

Observation
The egg white appeared as a viscous, clear,
yellowish liquid w/ some white particles
Viscous, clear, yellowish liquid with some
white particles clumping together
Filtrate: Viscous, clear, yellowish liquid
Residue: White clumps
Viscous, clear, yellowish liquid with clumps
Filtrate: Viscous, clear, yellowish liquid
Residue: White clumps
Cloudy liquid
Filtrate: Clear, yellowish liquid
Residue: Creamy substance
White crystals

Separate egg white from the yolk
Stir to break up the membrane
Filter through cheesecloth
Add acetic acid w/ constant stirring
Filter through cheesecloth
Add ammonium sulfate
Filter through filter paper
Air dry
Mass of filter paper

:

1.069 g

Mass of filter paper + crude egg albumin

:

9.326 g

Mass of crude egg albumin (actual yield)

:

8.257 g

Salting out is a process to precipitate proteins through the use of an aqueous solution of
high ionic strength. The solubility of the proteins is decreased due to the change of conformation
brought by the folding of the proteins to keep away hydrophobic functional groups from the polar
environment, letting the hydrophilic functional groups to interact with water. Charged amino
acids allow this process to occur because water is required to surround the molecule to remain
dissolved in the solution, however, the high ionic strength of the solution prevents the water from
supporting the proteins, thereby causing them to precipitate. (NSF & University of California,
2016)
In the experiment, the egg white was first stirred and filtered through a cheese cloth to
remove the egg membrane and any aggregates. The addition of the acetic acid is to further
precipitate and isolate other proteins through denaturation.

A crude sample of ovalbumin isolate was obtained after filtration and air drying which appeared as white crystals.7 since the aggregation occurred at this pH. 4.7 could be the IpH of casein since these aggregates show that the net charge of the proteins is zero.6 which is significantly near the determined value. PH 2. No repulsion from like-charged proteins occurs. the protein is predominantly negatively charged and the like-charged proteins exhibit repulsive forces thus. The IpH of casein determined from the experiment is 4.7 4.52% Table 4. the proteins are predominantly positively charged and there is also repulsion thus.7. the proteins are also water-soluble. therefore. mL) x 100% = (8. Furthermore. The same is also true with pH 6. The repulsion between negative charges caused the protein to also remain soluble in water.2. the crude egg ovalbumin was successfully separated from the egg white using ammonium sulfate precipitation. they are soluble in water.The increasing concentration of ammonium sulfate increases the ionic strength of the solution and causes more of the proteins to fold.are both at the ends of the Hofmeister series.7 6. the proteins tend to aggregate. thus. Test tube no. Below the IpH. with a high ability to solubilize protein structures. The yield of egg white albimun was calculated using the equation: %(w/v) = (mass of yield. Isoelectric precipitation of casein. Ammonium sulfate was used in the experiment due to its high solubility in water therefore allowing solutions of high ionic strength. It also decreases the availability of water to support the charges in the protein and cause them to precipitate.7 Observation Clear solution Cloudy solution Clear solution Proteins tend to precipitate at their isolectric pH (IpH) where their charge is zero.7.1. Other considerations may be the price of the substance and its availability. 1 2 3 pH 2. 2011) References: .7 could be below the IpH since the repulsion between the positively charged proteins caused the proteins to remain soluble in water.257 g / 30 mL) x 100% The value obtained is 27. This means that some of the proteins formed bigger aggregates that could be seen by the naked eye. g / volume. the solution appeared cloudy at pH 4. (Islamic University of Gaza) In the experiment. Furthermore. (Duong-Ly & Gabelli. the literature value for the IpH of casein is 4. Above the IpH. (Amrita Vishwa Vidyapeetham University. 2014) In the experiment. the NH4+ and SO4.

Chapter 7.eu Duong-Ly.org .net Islamic University of Gaza Faculty of Health Sciences. Isoelectric precipitation of proteins: Casein from milk. Salting out of Proteins Using Ammonium Sulfate Precipitation.Amrita Vishwa Vidyapeetham University. Methods in Enzymology volute 541. Retrieved from Researchgate. 2016. Retrieved from iugaza. Sandra.amrita.ps National Science Foundation and University of California. 2014. Retrieved from vlab. 2011. Retrieved from Libretexts. Precipitation of Proteins at Isoelectric Point.edu. Salting Out. Krisna and Gabelli.