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Experiment 5
Ebru AKHARMAN, Gebze Technical University, Turkey

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

fact that proteins precipitate differentially
The three dimensional(tertiary) structure from solution on the addition of species
of proteins is maintained by a number of such as neutral salt sor organic solvents. It
forces, mainly hyrdofobic interactions, should be stressed here that these methods
hydrogen bonds and sometimes disulphide precipitate native protein that has become
bridges. When we say that a protein is insoluble by aggregation.
denatured we mean that these bonds have
by some means been disrupted and that the Salt fractionation is ferquently carried out
protein chain has unfolded to give the using ammonium sulphate, sodium chloride,
insoluble, denetured protein. One of the sodium sulphate. As increasing salt is added
easiest ways to denature proteins in to a protein solution, so the salt ions are
solution is to heat them. However, different solvated by water molecules in the solution .
proteins will denature at different As the salt concentration increases, freely
temperatures. Proteins differ in the balance available water molecules that can solvate
of charged, polar and hydrophobic amino the ions become scarce. At this stage those
acids that they display on their surfaces. water molecules that have been forced into
Charged and polar groups on the surface are contact with hydrophobic groups on the
solvated by water molecules, thus making surface of the protein are the next most
the protein molecule soluble, whereas freely available water molecules (rather
hydrophobic residues are masked by water than those involved in solvating polar
molecules that are necessarily found groups on the protein surface, which are
adjacent to these regions. Since solubility is bound by electrostatic interactions anda re
a consequence of solvation of charged and far less easily given up ) and these are
polar groups on the surfaces of the protein, therefore removed to solvate the salt
it follows that, under the particular set of molecules, thus leaving the hydraphobic
conditions, proteins will differ in their 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 sulfate [2 4 ] to bring the solution to
large container of a given buffer or distilled saturation with respect to this salt. Use the
water. Thus small solute molecules freely same general procedure used for the
pass through the membrane, and after addition of ammonium sulfate. When the
several hours of stirring the equilibrium will added salt fails to dissolve, allow the
reach (the concentration inside and outside solution to stand for about 10 minutes and
the bag are the same). then fitler. Save the filtrate.
A proteins s multiple acid base
groups make its solubility Finally, using a third 3 ml sample of the
properties dependent on the filtered dilute egg white, add suficien solid
concentration of dissolved salts, the sodium chloride [] to bring the solution
polarity of the solvent, the pH, and to saturation with respect to this salt. Use
the temperature. the same general procedure used for the
addition of ammonium sulfate. When the
Solubility of a protein in aqueous added salt fails to dissolve, allow the
solution is a sensitive function of the solution to stand for about 10 minutes and
concentrations of dissolved salts. then fitler. Save the filtrate.
The salt concentration is expressed
At this point, you have three filtrates, each
in terms of the ionic strenght. of which is saturated with respect to
MATERIAL AND METHODS: particular salt and from each of which a
certain amount of the original protein has
3 Falcons been removed by precipation. You now
3 Tubes want to determine how effective these three
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 determination, you will need to remove the
Dialysis bag ammonium sulfate from your first filtrate.
Filter page So, the dialysis is maked for each salt
Obtain a 3 ml sample of 1/5 dilute egg
white. To a 3 ml sample of the filtered dilute RESULTS:
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 you used in regard to their abilities to
this, slowly, with stirring, add the solid precipitate proteins, you will need to
ammonium sulfate until it no longer goes
into solution. Be sure to allow adequate 1. the concentration of the protein in the
time after each increment of ammonium original filtered dilute egg white,
sulfate is addd to determine whether it will
dissolve. When the added salt fails to 2. the concentrations of protein in each of
dissolve, allow the solution to stand for the filtrates resulting from the salt
about 10 minutes and then fitler. Save the precipitations,
3. the volume change that occured during
To another 3 ml sample of the filtered dilute the dialysis of the filtrates.
egg white, add sufficient solid sodium
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.

Absorbance - Concentration Graph
1,6 1,57845

b y = 1,1148x
1,2 1,17845 R = 0,9792
o Seri 1
b 0,82545 Dorusal (Seri
a 1)
n 0,65995
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

Ccorrected = 0,522 x

Ccorrecte d = 0, 2245 mg / ml

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

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

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.

% protein precipitate 87
Seri 1
NaCl Na2SO4 (NH4)2SO4

Graph 2: % Protein Precipitation Graph

which case the solubility of the proteins
Proteins participate in many vital activities. increases. This is called salting. When a
The precipitation process used to separate large amount of neutral salt is added to the
the relevant protein from other proteins or protein solution, the water molecules
to concentrate the proteins is used in most around the hydrophobic groups, which are
of the purification processes of the proteins. usually found in the inner parts of the
For the purpose of the study, precipitation proteins, are removed by salt ions, in which
of proteins can be carried out in the fraction hydrophobic groups interfere with each
obtained by centrifugation and filtration or other and proteins precipitate. This is called
directly in the fermentation medium. The salting out. This is called salting out. In this
distribution of hydrophilic and hydrophobic experiment different salts were added to the
groups on the surface of the protein diluted egg white solution. The protein
molecule affects the solubility of the precipitation efficiencies of these salts were
proteins in various solvents. Using these determined by the following formulas. A bar
properties of the proteins, precipitation graph was generated with the obtained data.
processes are carried out in different forms According to this graph, the most efficient
according to the purpose. These are salt was determined to be ammonium
precipitation processes in pH, heat, organic sulfate.
solvents, organic polymers and high salt
concentrations. The precipitation of these RESOURCES:
salts is the most preferred of these
precipitation processes. The solubility of the
proteins in water is very low. When the precipitation/
medium is added with some neutral salt
(such as NaCl and (NH4)2SO4), the 2)
hydrophobicity of the proteins on the Di/Denaturation.html
surface or inside of the proteins increases
with the degree of interaction with water, in