Experiment 5-6: PROTEIN PRECIPITATION BY SALTS

Edanur Çakmak,152204032, Gebze Technical University

SUMMARY
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INTRODUCTION

The solubilities of lysozyme, alpha- salting in. effects of salts such as sodium
chymotrypsin and bovine serum albumin chloride [NaCl] on increasing the solubility
(BSA) were studied in aqueous electrolyte of proteins is often referred to as salting in
solution as a function of ionic strength, pH, (2).
the chemical nature of salt, and initial Some salts, such as high concentrations of
protein concentration. Compositions were ammonium sulfate, have general effects on
measured for both the supernatant phase solvent structure that lead to decreased
and the precipitate phase at 25 degrees C. protein solubility and salting out.when the
Salts studied were sodium chloride, sodium proteins precipitate at high concentration
sulfate, and sodium sulfate. Solubility can of salts it called Salting out (3).
be represented by the Cohn salting-out
equation. The effectiveness of the three salts Hofmeister Series
studied for precipitation were in the The effects of ions on biological and
sequence (NH4)2SO4 > Na2SO4 > sodium chemical processes in aqueous
chloride, consistent with the Hofmeister solution f usually depend on the particular
series (1). ions involved.
However, for alpha-chymotrypsin and Anions: SO42- > HPO42 ->
BSA, initial protein concentration affects acetate- > Cl- > NO3-
the apparent equilibrium solubility
When the solubility of proteins increased Cations: Mg2+ > Li+ >
in low concentration of salts it called Na+ = K+ > NH4+
Dialysis
Removal of salt molecules from the
isolated protein solution through a semi
permeable dialysis bag is called dialysis.
The salt molecules move from the more
concentrated solution (from inside the
dialysis bag) to the less concentrated
solution ( e.g. distilled water).
EXPERIMENTAL PROCEDURES
effective these three salts. You will need to
remove the ammonium sulfate from your
first filtrate so that dialysis is maked for each
salt solution.
RESULTS
To determine the effectiness of the salts,
you used in regand their abilities to
precipitate proteins, we should deterimine;
the concentration of the protein in the

-Obtain 3 ml sample of 1/5 dilute egg white.
To a 3 ml sample of the filtred dilute egg
white and sufficient solid ammonium sulfate
[Na2SO4] to bring the solution to saturation
with respect to this salt. To do this slowly
orginal filtreted dilute egg white, the
concentration of protein in each of the
filtrates resulting from the salt
precipitation, the value change that
occurred during the dialysis of the filtrates.

with stirring, add the solid ammonium

sulfate until it no longer goes into solution.
SALT Vbefore Vafter Abs Abs
-To another 3 ml sample of the filtred dilute (ml) Rep1 Rep2
egg white, add sufficient solid sodium NaCl 4ml 6,8 0,767 0,743
sulfate to bring the solution to saturation Na2SO4 4ml 6,6 0,554 0,503
(NH4)2SO4 4ml 6,9 0,368 0,36
with respect to this salt.
-Using a third 3 ml sample of the filtered
dilute egg white, add sufficient solid sodium
chloride to bring the solution to saturation
with respect to this salt.
-You have three filtrates, each of which is
saturated with respect yo particular salt
form each of which a certain amount of the
orginal protein has been removed by
precipitation. You want to determine how
 50
%protein precipitate 45
40
35
30 DISCUSSION
25
20 Firstly experiment, spectrophotometry was
15 used to determining maximum absorbance
10 for Methyl Red. It is important because if
5 maximum absorbance is found for
0 chemical, standard curve could be occurred
NaCl Na2SO4 (NH4)2SO4 much better. Our measured methyl red
Salts absorbance is 524 nm however it is 525 nm
for literature. These values are different
Figure 1: %protein precipitation graphs because a lot of factor that are environment
condition or error, could change results.
Many physical chemistry laboratory
courses fail to include an experiment
involving the determination of a
dissociation constant of a weak acid or
base; methyl red is a particularly good acid
for this purpose so that we prepared
different concentration of Methyl Red to
compare the connection between
absorbance and concentration. We saw that
if concentration increasing, absorbance
increase. The graphic was occurred to
show it by using least squares method. A
equation existed with holder value in the
shape of y=ax+b. Y is absorbance and X is
concentration. Also, unknown
concentration was found by making
standard curve.

REFERENCES
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