PROTEIN PRECIPITATION BY SALTS

Experiment 5
Ebru AKHARMAN, Gebze Technical University, Turkey

AIM:
Protein molecules carry out many important tasks in living systems. Most important, the
majority of proteins are quite specific about which task they perform. Protein structure is what
dictates this specificity, and the three-dimensional (tertiary) structure is particularly important.
When this specific three-dimensional structure is disrupted, the protein loses its functionality
and is said to have undergone denaturation. The interactions, such as hydrogen bonding , that
dictate the tertiary structure of proteins are not as strong as covalent chemical bonds. Because
these interactions are rather weak, they can be disrupted with relatively modest stresses. We
will use this property of proteins and thus,the proteins of egg white will precipitation with
different salts in this experiment. The precipitation capacities of salts will be obtained.

INTRODUCTION: solubilities. In particular, one exploits the

The three dimensional(tertiary) structure
of proteins is maintained by a number of
forces, mainly hyrdofobic interactions,
hydrogen bonds and sometimes disulphide
bridges. When we say that a protein is
denatured we mean that these bonds have
by some means been disrupted and that the
protein chain has unfolded to give the
insoluble, denetured protein. One of the
easiest ways to denature proteins in
solution is to heat them. However, different
proteins will denature at different
temperatures. Proteins differ in the balance
of charged, polar and hydrophobic amino
acids that they display on their surfaces.
Charged and polar groups on the surface are
solvated by water molecules, thus making
the protein molecule soluble, whereas
hydrophobic residues are masked by water
molecules that are necessarily found
adjacent to these regions. Since solubility is
a consequence of solvation of charged and
polar groups on the surfaces of the protein,
it follows that, under the particular set of
conditions, proteins will differ in their
fact that proteins precipitate differentially
from solution on the addition of species
such as neutral salt sor organic solvents. It
should be stressed here that these methods
precipitate native protein that has become
insoluble by aggregation.
Salt fractionation is ferquently carried out
using ammonium sulphate, sodium chloride,
sodium sulphate. As increasing salt is added
to a protein solution, so the salt ions are
solvated by water molecules in the solution .
As the salt concentration increases, freely
available water molecules that can solvate
the ions become scarce. At this stage those
water molecules that have been forced into
contact with hydrophobic groups on the
surface of the protein are the next most
freely available water molecules (rather
than those involved in solvating polar
groups on the protein surface, which are
bound by electrostatic interactions anda re
far less easily given up ) and these are
therefore removed to solvate the salt
molecules, thus leaving the hydraphobic
patches exposed. As the ammonium
sulphate concentrarion increases, the Different type of salts such as ammonium
hydrophobic surfaces on the protein are sulfate and sodium sulfate are widely used
progressively exposed. Thus revealed, these to precipitate out proteins.
hydrophobic patches cause proteins to
aggregate by hydrophobic interaction, Ammonium sulfate is the most widely used
resulting in precipitation. The first proteins salt for the precipatation of proteins as it is
to aggregate are therefore those with most highly soluble, inexpensive, available in
hydrophobic residues. highest purity level, does not change the
Clearly the aggregates formed are made of protein solution to extreme pHs and in most
mixtures of more than one protein. case it does not denature proteins.
Indivudual identical molecules do not seek
out each other, but simply bind to another
adjacent molecule with an exposed
hydrophobic patch. However, many proteins
are precipitated from solution over a
narrow range of salt concentrations, making
this suitably simple procedure for enriching
the proteins of interest.
The solubility of a protein at low ion
concentrations increases as salt is added, a
phenomenon called "salting in". The
additional ions shield the protein's multiple
ionic charges, thereby weakening the
attractive forces between individual protein
molecules (such forces can lead to
aggregation and precipitation).
However, as more salt is added, particularly
with sulfate salts, the solubility of protein
Image 1: The Image of Dialysis
again decreases. This "salting out" effect is
primarily a result of the competition Dialysis is one of the common operations in
between the added salt ions and the other biochemistry to separate dissolved
dissolved solutes (protein molecules) for molecules by passing through a semi-
molecules of solvent (water). permeable membrane according to their
molecular dimensions. Semi-permeable
There is a series of the relative effectiveness membrane is containing pores of less than
of different anions in the salts used for macromolecular dimensions. These pores
protein precipitation. This is referred to as allow small molecules, such as those of
the Iyotropic of Hofmeister series. solvents, salts, and small metabolites, to
diffuse across the membrane but block the
Anions: passage of larger molecules. Cellophane

> > > >

(cellulose acetate) is the most commonly
Cations: used dialysis material although many other
+
> = > >
+ + + +
substances such as nitrocellulose and
Table 1: Hofmeister Series Table collodion are similarity employed. So,
dialysis is a method in which an aqueous
solution containing both macromolecules
and very small molecules which are placed
in a dialysis bag which is in tern placed in a
large container of a given buffer or distilled
water. Thus small solute molecules freely
pass through the membrane, and after
several hours of stirring the equilibrium will
reach (the concentration inside and outside
the bag are the same).
A proteins s multiple acid base
groups make its solubility
properties dependent on the
concentration of dissolved salts, the
polarity of the solvent, the pH, and
the temperature.
Solubility of a protein in aqueous
solution is a sensitive function of the
concentrations of dissolved salts.
The salt concentration is expressed
in terms of the ionic strenght.
MATERIAL AND METHODS:
3 Falcons
3 Tubes
4 g ammonium sulfate[(4 )2 4 ] saltsa re in regard to precipating egg white
2 g solid sodium sulfate [2 4 ] proteins. However, before you perform tha
2 g solid sodium chloride [] assays that are necessary to make this
9 ml 1/5 diluted egg white
Dialysis bag
Filter page
Obtain a 3 ml sample of 1/5 dilute egg
white. To a 3 ml sample of the filtered dilute
egg white, and sufficient solid ammonium
sulfate [2 4 ] to bring the solution to To determine the effectiveness of the salts
saturation with respect to this salt. To do
this, slowly, with stirring, add the solid
ammonium sulfate until it no longer goes
into solution. Be sure to allow adequate
time after each increment of ammonium
sulfate is addd to determine whether it will
dissolve. When the added salt fails to
dissolve, allow the solution to stand for
about 10 minutes and then fitler. Save the
filtrate.
To another 3 ml sample of the filtered dilute
egg white, add sufficient solid sodium
sulfate [2 4 ] to bring the solution to
saturation with respect to this salt. Use the
same general procedure used for the
addition of ammonium sulfate. When the
added salt fails to dissolve, allow the
solution to stand for about 10 minutes and
then fitler. Save the filtrate.
Finally, using a third 3 ml sample of the
filtered dilute egg white, add suficien solid
sodium chloride [] to bring the solution
to saturation with respect to this salt. Use
the same general procedure used for the
addition of ammonium sulfate. When the
added salt fails to dissolve, allow the
solution to stand for about 10 minutes and
then fitler. Save the filtrate.
At this point, you have three filtrates, each
of which is saturated with respect to
particular salt and from each of which a
certain amount of the original protein has
been removed by precipation. You now
want to determine how effective these three
determination, you will need to remove the
ammonium sulfate from your first filtrate.
So, the dialysis is maked for each salt
solutions.
RESULTS:
you used in regard to their abilities to
precipitate proteins, you will need to
determine
1. the concentration of the protein in the
original filtered dilute egg white,
2. the concentrations of protein in each of
the filtrates resulting from the salt
precipitations,
3. the volume change that occured during
the dialysis of the filtrates.
 Absorbance Absorbance Absorbance Absorbance Avg.- Blank
Concentration(mg/ml) Rep1 Rep2 Avg. Avg.
A 1,5 (mg/ml) 1,6330 1,5680 1,6005 1,57845
B 1 (mg/ml) 1,2020 1,1990 1,2005 1,17845
C 0,75 (mg/ml) 0,8540 0,8410 0,8475 0,82545
D 0,5 (mg/ml) 0,6900 0,6740 0,6820 0,65995
E 0,25 (mg/ml) 0,4010 0,4030 0,4020 0,37995
F 0,125 (mg/ml) 0,2220 0,2380 0,2300 0,20795
Blank G 0 (mg/ml) 0,0225 0,0216 0,0221 0,00000
Egg White H X (mg/ml) 1,2350 1,2400 1,2375 1,21545
Table 2: Absorbance Table

This table is obtained from different concentration values. The absorbance of these
concentrations values are measured with Bradford assay. The absorbance of blank is removed
from other absorbance values. Thus, effect of buffer is removed. These datas are used to create
absorbance concentration graph.

Absorbance Absorbance Avg.- Blank

SALT Vbefore (ml) Vafter (ml) Abs Rep1 Abs Rep2 Avg. Avg.
NaCl 3,0 ml 7,0 ml 0,601 0,606 0,6035 0,58145
3,0 ml 7,1 ml 0,440 0,445 0,4425 0,42045
(NH4)2SO4 3,0 ml 7,2 ml 0,356 0,359 0,3575 0,33545
Table 3: Absorbance of Salts Table

This table shows absorbances of different salt solutions. These datas are used for calculating %
protein precipitation.

1,8
Absorbance - Concentration Graph
1,6 1,57845

1,4
A
b y = 1,1148x
1,2 1,17845 R = 0,9792
s
o Seri 1
1
r
b 0,82545 Dorusal (Seri
0,8
a 1)
n 0,65995
0,6
c
e 0,4 0,37995

0,2 0,20795

0 0
0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6
Concentration (mg/ml)
Graph 1: Absorbance Concentration Graph
Equation 1: Equation 2:

= (/ ) % protein precipitation

Bradford protein assay is used to =

determine the protein concentration of
each of samples.

For original diluted egg white; For ;

y = 1,1148 X ( y = 1,21545) y = 1,1148 X ( y = 0,58145 )

1,21545 0,58145
Coriginal = Cobserved =
1,1148 1,1148

Coriginal 1,0903 mg / ml Cobserved 0, 522 mg / ml

3
Ccorrected = 0,522 x
7

Ccorrecte d = 0, 2245 mg / ml

(1,09030,2243 )
= 100 1,0903
% protein precipitated for NaCl is
79,42.

For ; For (NH4)2SO4 ;

y = 1,1148 X ( y = 0,42045) y = 1,1148 X ( y = 1,21545)

0,42045 0,33545
Cobserved = Cobserved = 1,1148
1,1148

Cobserved 0, 377 mg / ml Cobserved 0, 301 mg / ml

3 3
Ccorrected = 0, 377 x Ccorrected = 0, 301 x
7 7

Ccorrected = 0, 1621 mg / ml Ccorrected = 0, 129 mg / ml

(1,09030,1621 ) (1,09030,129 )
= 100 = 100
1,0903 1,0903

% protein precipitated for Na2 SO4 is % protein precipitated for (NH4)2SO4

85,16. is 88, 17.

Table 4: Calculates Table

According to this datas, a bar graph is created.

 89
% protein precipitate 87
85
83
81
79
77
75
NaCl
Graph 2: % Protein Precipitation Graph
DISCUSSION:
Proteins participate in many vital activities.
The precipitation process used to separate
the relevant protein from other proteins or
to concentrate the proteins is used in most
of the purification processes of the proteins.
For the purpose of the study, precipitation
of proteins can be carried out in the fraction
obtained by centrifugation and filtration or
directly in the fermentation medium. The
distribution of hydrophilic and hydrophobic
groups on the surface of the protein
molecule affects the solubility of the
proteins in various solvents. Using these
properties of the proteins, precipitation
processes are carried out in different forms
according to the purpose. These are
precipitation processes in pH, heat, organic
solvents, organic polymers and high salt
concentrations. The precipitation of these
salts is the most preferred of these
precipitation processes. The solubility of the
proteins in water is very low. When the
medium is added with some neutral salt
(such as NaCl and (NH4)2SO4), the
hydrophobicity of the proteins on the
surface or inside of the proteins increases
with the degree of interaction with water, in
Seri 1
Na2SO4 (NH4)2SO4
Salts
which case the solubility of the proteins
increases. This is called salting. When a
large amount of neutral salt is added to the
protein solution, the water molecules
around the hydrophobic groups, which are
usually found in the inner parts of the
proteins, are removed by salt ions, in which
hydrophobic groups interfere with each
other and proteins precipitate. This is called
salting out. This is called salting out. In this
experiment different salts were added to the
diluted egg white solution. The protein
precipitation efficiencies of these salts were
determined by the following formulas. A bar
graph was generated with the obtained data.
According to this graph, the most efficient
salt was determined to be ammonium
sulfate.
RESOURCES:
1)https://bitesizebio.com/5924/the-ins-and-
outs-of-protein-concentration--protein-
precipitation/
2)http://www.chemistryexplained.com/Co-
Di/Denaturation.html
3)https://eng.umd.edu/~nsw/ench485/lab
6a.htm#Discussions