Practical No.

06 : Separation of proteins using (NH4)2SO4 precipitation

Objective : To gain experience in separation and purification techniques for proteins

Introduction : The solubility of protein depends on, among other things, the salt
concentration in the solution. At low concentrations, the presence of salt stabilizes the various
charged groups on a protein molecule, thus attracting protein into the solution and enhancing the
solubility of protein. This is commonly known as salting-in. However, as the salt concentration is
increased, a point of maximum protein stability is usually reached. Further increase in the salt
concentration implies that there is less and less water available to stabilize protein. Finally, protein
starts to precipitate when there are not sufficient water molecules to interact with protein molecules,
this phenomenon of protein precipitation in the presence of excess salt is known as salting-out.

Many types of salts have been employed to effect protein separation and purification through salting-
out. Of these salts, ammonium sulfate has been the most widely used chemical because it has high
solubility and is relatively inexpensive.

Materials:

Protein solutions, solid ammonium sulfate, saturated ammonium sulfate solution, pipettes and pipette
fillers, Beakers, Magnet stirrer, Centrifuge tubes, Centrifuge

Procedure:

Ammonium sulfate ((NH4)2SO4) can be added

- as a solid: see Table 1 for amounts to be added to reach a saturation level

- from a saturated (= 100%, w/v) solution

Using (NH4)2SO4 Solid

Concentration series was prepared according to the following amounts of (NH4)2SO4.

Table 01: Different concentrations of (NH4)2SO4 mixed with legume seeds

(NH4)2SO4 0-30% 30-45% 45-60% 60-75% 75-90%

Amount for 1L (g) 176 92 97 103 110
Amount for 1.5ml 0.264 0.138 0.146 0.155 0.165
(g)

(NH4)2SO4 solid was weighed to separate 1.5ml as above. Then 1.5ml of Legume seeds (Chick-pea)
serum was added to 0-30% concentration tube and mixed immediately. This was kept on ice for a ½
hour and centrifuged 10,000rpm for 10minutes. Then 1.5ml of supernatant was transferred to next
tube which concentration was 30-45%, mixed and kept on ice as above. This procedure was followed
for all. 0-30% tube was again centrifuged and discarded the supernatant. The pellet was dissolved in
500µl of 1x PBS

Using Saturated (NH4)2SO4 solution (100%)

700µl of Albumin serum and 300 µl of saturated (NH4)2SO4 were measured in to a test tube to 0-
30% (NH4)2SO4 saturation. This was kept on ice for a ½ hour and centrifuged 10,000rpm for
10minutes. Supernatant (S1) and pellet (P1) were collected separately. 214 µl saturated (NH4)2SO4
was added to 786 µl of supernatant (S1) to 30-45% (NH4)2SO4 saturation. This was kept on ice for a
½ hour and centrifuged 10,000rpm for 10minutes. Supernatant (S2) and pellet (P2) were collected
separately. This procedure was followed for all, as given in the table 2. 0-30% tube was again
centrifuged and discarded the supernatant. The pellet was dissolved in 500µl of 1x PBS

Table 02: Different concentrations of (NH4)2SO4 mixed with casine protein solution

(NH4)2SO4 0-30% 30-45% 45-60% 60-75% 75-90%

Concentration
Supernatant 700 786 727 625 400
Volume(µl)
Saturated (NH4)2SO4 300 214 273 375 600
volume(µl)
Total Volume(µl) 1000 1000 1000 1000 1000

Observations:

Table 03: Remark of protein precipitation from (NH4)2SO4 Solid

(NH4)2SO4 0-30% 30-45% 45-60% 60-75% 75-90%

Amount for 176 92 97 103 110
1L (g)
Amount for 0.264 0.138 0.146 0.155 0.165
1.5ml (g)
Remark on Considerable significant Insignificant Insignificant Insignificant
precipitation Protein Protein precipitation precipitation Precipitation
Precipitation Precipitation
occurred occurred
Table 04: Remark of protein precipitation from Saturated (NH4)2SO4 solution (100%)

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

(NH4)2SO4 0-30% 30-45% 45-60% 60-75% 75-90%
Concentration
Supernatant 700 786 727 625 400
Volume(µl)
Saturated 300 214 273 375 600
(NH4)2SO4
volume(µl)
Total Volume(µl) 1000 1000 1000 1000 1000
Remark on Considerable significant Slightly Insignificant Insignificant
precipitation Protein Protein significant precipitation Precipitation
Precipitation Precipitation Protein
occurred occurred Precipitation
occurred

Sample 01 Sample 02 Sample 03 Sample 04 Sample 05

Figure 01: Different amounts of precipitates in different sample of chick- pea protein solutions

Sample 01 Sample 02 Sample 03 Sample 04 Sample 05

Figure 02: Different amounts of precipitates in different sample of casein protein solutions
Conclusion: Proteins in Aqueous solutions are heavily hydrated, and with the addition of salt ,the
water molecules become more attracted to the salt than to the protein due to the higher charge .This
competition for hydration is usually more favorable towards the salt, which leads to interaction
between the proteins, resulting in aggregation and finally precipitation. When the (NH4)2SO4
concentration is gradually increased, the ionic strength of the solutions is also increased accordingly.
So the rate of agglomeration of the proteins is proportional to the concentration of the (NH4)2SO4
solutions.

Discussion:

In protein purification, it is important to adopt procedures that do not cause denaturation of proteins,
especially the protein of interest. The choice of purification methods is also influenced by factors
such as how the purified protein is to be used in studies, the quantity of the purified protein needed,
and the cost of the materials and reagents used in the purification. A purification step that may
denature purified protein is not suitable for studies of its biological properties, but may be suitable
for the determination of its primary structure, subunit size, etc. The purification protocols for
obtaining a microgram level of purified protein may be different from those that yield larger
quantities of purified protein. The cost of ligands used for immobilization of matrix and for elution
of a bound protein in affinity chromatography may be limiting factors for large-scale purification. A
protein may be purified by a single step (for example, affinity chromatography), or by a combination
of several steps (for example, salt fractionation, ion exchange, gel filtration, etc.). In general, anion-
exchange chromatography is employed for the purification of an acidic protein. Similarly, for the
purification of a basic protein, cation-exchange chromatography is the better choice. Reverse-phase
chromatography is suitable for a family of active proteins of similar charge.

Precipitation of proteins by increasing the ionic strength of the solution is also knows as protein
salting out. Salting out is dependent on the hydrophobicity on the surface of the protein.
Hydrophobic regions are enriched in the hydrophobic amino acids, phenylalanine, tyrosine,
tryptophan, leucine, isoleucine, methionine and valine. Proteins with more hydrophobic regions will
aggregate and precipitate before those with smaller and fewer hydrophobic regions, thus resulting in
fractionation. Salting out is usually performed at 4C to decrease the risk of inactivation.

Salting out proteins from a crude extract using ammonium sulfate is a convenient purification step.
Salts affect the electrostatic and non-polar properties of proteins in a reversible manner. At
concentrations above 0.2M, salts not only neutralize the electrostatic forces on the protein surface but
also affect the three dimensional structure of proteins, making them less soluble.

Salts, such as ammonium sulfate, have the tendency to disrupt the water structure, increase the water
surface tension and increase the hydrophobic effect in the solution (i.e. decrease the solubility of
non-polar molecules) and promote protein aggregation by association of hydrophobic surface.
Ammonium sulfate is the most frequently used salt for salting-out experiments and can precipitate
~70% of proteins in a complex sample.

The solubility of proteins varies according to the ionic strength of the solution, and hence according
to the salt concentration. Two distinct effects are observed: at low salt concentrations, the solubility
of the protein increases with increasing salt concentration (i.e. increasing ionic strength), an effect
termed salting in. As the salt concentration (ionic strength) is increased further, the solubility of the
protein begins to decrease. At sufficiently high ionic strength, the protein will be almost completely
precipitated from the solution (salting out).

Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful
procedure to assist in the purification of a given protein. The commonly used salt is ammonium
sulfate, as it is very water soluble, forms two ions high in the Hofmeister series, and has no adverse
effects upon enzyme activity. It is generally used as a saturated aqueous solution which is diluted to
the required concentration, expressed as a percentage concentration of the saturated solution (a 100%
solution).

The precipitated protein is then removed by centrifugation and then the ammonium sulfate
concentration is increased to a value that will precipitate most of the protein of interest whilst leaving
the maximum amount of protein contaminants still in solution. The precipitated protein of interest is
recovered by centrifugation and dissolved in fresh buffer for the next stage of purification.

This technique is useful to quickly remove large amounts of contaminant proteins, as a first step in
many purification schemes. It is also often employed during the later stages of purification to
concentrate protein from dilute solution following procedures such as gel filtration.

Tris is a chemical with basic properties, having a pKa of 8.1. It can be used to buffer solutions from
drastic pH changes, keeping them in the pH range of 7.0 to 9.0.

Assignment:

1. What is the theory behind the precipitation of proteins using ammonium sulphate?

Salting out proteins from a crude extract using ammonium sulfate is a convenient purification
step. Ammonium sulfate precipitation is a method used to purify proteins by altering their
solubility. Ammonium sulfate is commonly used as its solubility is so high that salt solutions
with high ionic strength are allowed.

Many cytosolic proteins are water soluble and their solubility is a function of the ionic
strength and pH of the solution. The commonly used salt for this purpose is Ammonium
 Sulphate, Due to its high solubility even at lower temperatures. Proteins in Aqueous solutions
are heavily hydrated, and with the addition Of salt ,the water molecules become more
attracted to the salt Than to the protein due to the higher charge .This competition For
hydration is usually more favorable towards the salt, which leads to interaction between the
proteins, resulting in aggregation and finally precipitation. The precipitate can then be
collected by Centrifugation and the protein pellet is re-dissolved in a low salt buffer. Since
different proteins have distinct characteristics, it is often the case that they precipitate (or salt
out) at a particular concentration of salt.

2. Describe an appropriate procedure for the quantification of the proteins in the sample?

Ultraviolet Absorption methods

In ultraviolet (UV) absorption methods, proteins are measured directly without the addition
of any reagents. Proteins have two absorption maxima: 280 nm and 200 nm. In absorption
spectroscopy, an electron absorbs photons. Photons with lower energy levels have longer
wavelengths, and thus electrons that are excited at 280 nm have absorbed less energy than
those at 200 nm. Electrons that are excited at 280 nm require less energy because they lie
within the aromatic rings, which stabilize the excited state due to resonance.

Absorbance at 280 nm (A280)

The quantization of protein by this method can only be applied to pure protein. Nonetheless,
absorbance is widely used for monitoring purification progress and for generating a protein
elution profile during column chromatography. For A280, the amino acids containing
aromatic rings, such as tryptophan, tyrosine, phenylamine, and histidine, are involved. The
method is relatively sensitive, being able to measure protein concentrations as low as 10
mgcm-3, and, unlike colorimetric methods, is non-destructive, i.e. having made the
measurement, the sample in the cuvette can be recovered and used further. This is particularly
useful when one is working with small amounts of protein and cannot afford to waste any.
However, the method is subject to interference by the presence of other compounds that
absorb at 280 nm.

Determination of protein concentration using A280

For an unknown protein or protein mixture, the following formula can be used to obtain a
rough estimate of protein concentration. Using this procedure, a protein of 20 µg/ml to 3
mg/ml can be measured.

Concentration (mg/ml) = A280/path length in cm

 3. Describe the available techniques for further purification of the sample?

Purification steps divide the total protein in the crude extract into several fractions, each of
which is then assayed for activity and protein content. A fraction with high specific activity
and purification-fold dictates the success of its purification step. Specific activity is defined
as the total activity per milligram of protein per milliliter in a fraction.

The Protein purification series allows protein fractionation using protein properties that are
affected by changing the pH and ionic strength of the protein solutions. The use of acid
and/or salt fractionations concentrates and enriches proteins into defined fractions, depending
on their precipitation at differing pH and salt concentrations. Adjusting the pH has been used
as a simple and efficient way to precipitate proteins. Proteins have their lowest solubility at
their isoelectric point. When the pH is gradually changed the pH of the solution passes
through the isoelectric point of some proteins, causing some proteins to precipitate.
Isoelectric precipitation is often used to precipitate unwanted proteins rather than to
precipitate the protein of interest.

Protein fractionation utilizes the varied properties of proteins to separate a complex biological
sample into more basic, enriched and concentrated samples. There are numerous properties
of protein that can be utilized to fractionate proteins, including size, shape, sedimentation
velocity, ability to bind to various ionic groups, affinity for substrates or pseudo-substrates,
solubility, stability, and many more. Basically fractionation of a protein of interest can use
any protein property that differs from unwanted proteins. For the fractionation of a protein of
interest from a complex biological sample, numerous fractionation properties are routinely
used.

Polyethylene glycol

- neutral, non-denaturating compound

- low heat of solution
- steric exclusion mechanism (binds water)

Protamine sulfate

- small, basic proteins from sperm(many Arg and Lys residues)

- precipitation of large protein complexes (ribosomes), DNA, RNA by complexation
- precipitation is concentration dependent
 Apolar solvents (acetone, alcohol)

- low temperature (- 5 OC)

- denaturation of proteins

Trichloroacetic acid

- 6% (w/v) final concentration

- unfolds/denaturates protein

References:

1. Walker,J, ‘Protein structure, purification, characterisation and function analysis’, in Keith

Wilson & John Walker (ed.) 1997, Principles and techniques of biochemistry and
molecular biology,7th edn, Cambridge University Press, pp. 300-51.
2. John M. Walker (ed) 1996, The protein protocols handbook, Humana Press, New Jersey.
3. Keith Wilson & John Walker (ed.) 1997, Principles and techniques of biochemistry and
molecular biology, 7th edn, Cambridge University Press.
4. Amersham Pharmacia Biotech AB 1999, Protein purification handbook, AB edn.
5. Heide, K. and Schwick, H. G. (1978) Salt fractionation of immunoglobulins, in Handbook
of Experimental Immunology, 3rd ed. (Weir, D. M., ed.), chap. 7. Blackwell Scientific,
Oxford, UK
6. Gladyshev, V. N., Jeang, K. T., Wootton, J. C., & Hatfield, D. L. (1998). A new human
selenium-containing protein. Purification, characterization, and cDNA sequence. The
Journal of Biological Chemistry, 273(15), 8910–8915.