Concentration

&
Purification of Target
Product by Precipitation

THE FIRST STEP OF PURIFICATION

(CAPTURE STATE)
ISOLATION, CONCENTRATION &
STABILISATION OF TARGET
MOLECULE
MOSTLY USED METHODS OF CONCENTRATION

1. Freeze-drying, also known as lyophilisation or

lyophilization or cryodesiccation
2. Dialysis by particular cut-off membrane
3. Ultrafiltration by particular cut-off membrane
4. Centrifugal Ultrafiltartion by using membrane
5. Concentration/fractionation by salting out
 PRECIPITATION
What is Precipitation ?

Precipitation involves the conversion of the soluble solutes into insoluble

solids (precipitate), which subsequently separated from the liquid by physical

methods of separation such as filtration or centrifugation.

 Now the solubility of proteins and the precipitations are dependent on the

following factors :
 Advantages of Protein Precipitation Method

1. Concentration of the desired product i.e. reduction in

the volume by a factor of 10-50 times
2. Rapid separation with simple methodology

3. Well established method for the recovery of bulk

products
4. The method is often used in the preliminary stages

5. Biological products or molecules even at very low

concentration, is captured
Examples :

(I) Fractionation of human blood plasma remains in use

even after 60 years.

(II) IgG and albumin of about 99% purity are obtained

from human blood
 Precipitation Based On

Solvent property modification Solute property modification

1. pH change (Isoelectric precipitation) 1. Selective interaction with metals,
polyelectrolytes or affinity reagents
2. Ionic strength change (Salting out)

3. Organic solvent mediated precipitation 2. Selective denaturation to precipitate

unwanted proteins by:
4. Precipitation by non-ionic polymer
(a) pH denaturation
(b) Thermal denaturation
(c) Organic solvent denaturation
 PRINCIPLES OF SOLVATION AND
PRECIPITATION OF PROTEINS
Proteins have definite molecular weight, size, shape, electrical
properties, and amino acid composition.

A typical globular protein in aqueous solution exhibits a non-

uniform distribution of surface positive and negative charges.

 The surface also exhibits hydrophilic as well as hydrophobic

regions.
 The solubility of the protein is determined by the interactions
between various regions with the surrounding water molecules.
 The solubility of protein solutions can be considered on the basis of their colloidal
nature.
 In physiological conditions i.e. in cellular fluids of ionic strength of 0.15  0.2M, most of
the proteins exist in soluble form.
Proteins in their natural condition generally exhibit a net –ve charge on the surface and
attract +ve ions to form the so-called Stern layer of counter ions close to the protein
surface.
 The Stern layer is surrounded by a diffuse Guoy-Chapman layer of mobile counter ions.

Stern Layer
(Primary)

+ +
+  + 
+ + 
+ + 
+ + 
+  +
+  +
+ +
+  +
+
+
Guoy-Chapman
Layer (Outer)
Hydrophobic
Patches

Figure 1: The surface of a protein in aqueous environment

Stability Of The Protein-electrolyte Colloid

 Due to the balance between the attractive and repulsive forces

between colloidal particles not allowing them to form aggregates.

 The Stern layer by controlling the effective thickness of the more

diffuse outer layer i.e. Guoy-Chapman layer determines the
stability of the colloid.

 The thickness of the double layer can be reduced by changing

the solvent characteristics, particularly the ionic strength and the
dielectric constant or by changing the protein surface
characteristics thereby bringing about a decrease in the solubility
of the protein.
MAJOR FORCES
 The major forces within a polypeptide chain and
between chains are ion-ion, ion-dipole (including
hydrogen bonds), dipole-dipole, and hydrophobic
interactions.

 Salt Ions in the medium may competitively disrupt ionic

interactions in the polypeptide.
 Isoelectric Precipitation
 By, changing the pH of the protein solution, the
ionization of the weak acidic and basic amino acid side
chains of a protein is affected resulting in a net zero
charge on the protein at a certain pH value called its
isoelectric point (pI) or isoelectric pH.

 When the pH of the protein solution at low ionic

strength is adjusted to a value equal to its pI, the
solubility of the globular protein decreases drastically
over a narrow pH range and it tends to precipitate out.
 Isoelectric Precipitation

Protein titration curves

Figure 4. The pI is the pH at which there is no net charge on the

protein. At lower pH, there are more positive charges in the
environment and therefore, the protein has an increased cationic
character. The reverse is true at pH above the pI.
 Isoelectric Precipitation
Effects of Ionic strength come into play at low salt
concentrations (0 0.2M).
At low ionic strength, protein solubility is at its minimum at
the proteins pI.
At this pH, intramolecular electrostatic forces between
oppositely charged side chains are at a maximum, protein
conformation is maximally tightened and protein hydration is
least.
On either side of the pI, titration of ionisable groups leads to
a lessening of intramolecular ionic interactions gives a more
relaxed conformation of protein and protein hydration and
solubility are increased.
 Isoelectric Precipitation

The following equation is called

Cohn equation : Log S =   kI
Where, S is the solubility of the protein
, is a constant dependent on pH
Solubility

k, is the salting out constant

I, is the ionic strength

n
I = ½  CiZi2
i

C is the concentration of the ion

Z is the charge on the ion

Figure 2: Solubility of globular proteins as a function of pH.

e.g. soy protein, casein, gelatin, collagen.

Concentration/Fractionation by Salting-Out

Salting out using ammonium sulfate is one of the

classical methods and widely used for the
fractionation of proteins, but it is not a highly
discriminating method and it is unusual to get a
pure fraction, using only this method.
It is rather used as an inexpensive way of
concentrating a protein extract, while leaving
protein and non-protein material in solution, and
any purification with respect to the target product
is generally regarded as a bonus.
 Salt Mediated Precipitation
Addition of low concentrations of salt causes a similar
weakening of intramolecular ionic bonds, with similar
consequences of more relaxed protein structure and greater
solubility. Addition of salt and altering of the pH, away from the
pI, have similar, and additive, effects. The increase in solubility
of protein upon addition of modest amounts of salt is known as
“salting in”.

“Salting in” of proteins the interaction of pH and ionic strength

What is salting-in?
At low salt concentration, the
presence of salt stabilizes the various
charged groups on a protein molecule,
thus attracting protein into the solution
(Pushing) and enhancing the solubility of
protein. This is commonly known as
salting-in.
What is salting-out ?
* However, as the salt concentration is increased,
a point of maximum protein solubility is usually
reached.
** Further increase in the salt concentration
implies that there is less and less water available
to solubilize protein.
*** Finally, protein starts to precipitate out when
there are not sufficient water molecules to
interact with protein molecules (Pulling). This
phenomenon of protein precipitation in the
presence of excess salt is known as salting-out.
 Salt Mediated Precipitation

 Different lines represent different proteins.

 By using the appropriate concentration range of the given
salt, we can precipitate the protein of interest, preferentially
form a mixture of proteins.

 The slope of the salting out curve is a function of the

protein and salt involved, but it is not a function of the pH and
temperature.

 Also, as the molecular weight of the protein increases, the

amount of salt required for precipitation decreases.
 Salt Mediated Precipitation

• Protein solubility is a complex function of the

physiochemical nature of the proteins: pH, temperature and
the concentration of the salt used. It also depends on
whether the salt is Kosomtropic (stabilizes water structure)
or Chaotropic (disrupts water structure).

• At low concentrations of salt, solubility of the proteins

usually increases (salting in). But at high concentrations of
salt, the solubility of the proteins drop sharply (salting out).
 Hoffmeister Series

 There are series of the relative effectiveness of different

anions and cations in the salts used for protein precipitation.
 Hofmeister series
The effects of these changes were first worked out by Franz Hofmeister, who
studied the effects of cations and anions on the solubility of proteins.

Hofmeister discovered a series of salts that have consistent effects on the

solubility of proteins and (it was discovered later) on the stability of their
secondary and tertiary structure. Anions appear to have a larger effect than
cations.

The order ofanions is usually given as:

The order of cations is usually given as:

Kosmotropy. At concentrations above 0.2 M the sulfate
ion acts as a Hofmeister kosmotrope, i.e. it stabilises
protein structure, and concomitantly reduces its
solubility. The effect of a kosmotrope, in stabilising
protein structure, can be described by the reaction:-

Kosmotropes may be described as “pushing” if they act

on the left of this reaction and “pulling” if they act on the
right.
Sulfate can act as a pulling kosmotrope by virtue of its interaction with protein
cationic sites.
1. Precipitation of proteins is usually promoted at pH values below the pI, where
the protein has a maximal number of cationic sites.
2. Sulfate ion is divalent, and so can bind to more than one cationic site at a
time, and that it has a tetrahedral structure, with four oxygen atoms that can
hydrogen bond to multiple sites on the protein.

Sulfate also acts as a pushing

kosmotrope by virtue of its
extraordinary hydration. By
virtue of its hydration, the
sulfate ion can act as a
dehydrating agent

The effect of pH on the salting out of a protein by ammonium sulfate.

 Ammonium Sulfate is Widely Used

¶ Many types of salts have been employed to effect

protein separation and purification through salting-
out.
¶ Ammonium sulfate has been the most widely used
salts because it has high solubility and is relatively
inexpensive.
Ammonium Sulfate is Widely Used
The sulfate ion has been viewed in a number of ways, regarding
how it salts out proteins:

1) Ionic strength effects,

2) Kosmotropy,
3) Exclusion-crowding,
4) Dehydration,
5) Binding to cationic sites, especially when the protein has a
net positive charge (denoted ZH+)

All of these may play a role, depending upon the salt

concentration and the pH-dependent charge on the protein.
Scopes Equation for Salting-Out

 The amount of ammonium sulphate to be added to reach a

required molar concentration M2 from an initial concentration M1
has been given by Scopes as:

g = 533(M2 –M1)/(4.05 - 0.3M2)

Where g is the amount (in grams) of ammonium sulphate required.

The factor 533 represents the solubility of the salt in grams at 200C
in one litre of water to make a saturated solution.

 Another equation of Scopes gives the quantity of ammonium

sulphate to be added for reaching a percentage saturation of S2
from an initial percentage saturation S1 as:

g = 533 (S2 – S1)/(100 - 0.3S2)

At high salt concentrations, the solubility is given by the following
empirical expression due to Cohn.
log S =  - KsI
Where, S is the solubility of the protein (g/l),
„‟ is a constant (function of protein type, pH and temperature of protein solution)
„Ks‟ is (slope of the curve) the salting out constant (slightly function of nature of
protein),

and „I‟ is the ionic strength of the salt.

Here you could plot the log of protein solubility versus the salt concentration, and it
would look like this:
Solubility of a
typical
protein vs
concentration
of ammonium
sulfate.
 a b c d

Log S

Salt concentration
a, fibrinogen (340 kDa); b, haemoglobin (68 kDa);
c, serum albumin (66 kDa); d, myoglobin (17 kDa)
Energetics involved in salting out
Salting out is a spontaneous process when the right concentration of the salt

is reached in solution. The hydrophobic patches on the protein surface

generate highly ordered water shells. This results in a small decrease in

enthalpy, ΔH, and a larger decrease in entropy, ΔS, of the ordered water

molecules relative to the molecules in the bulk solution.

Energetics involved in salting out
The overall free energy change, ΔG, of the process is given by
the Gibb’s free energy equation:
ΔG = ΔH − TΔS.
ΔG = Free energy change,
ΔH = Enthalpy change upon precipitation
ΔS = Entropy change upon precipitation
T = Absolute temperature
When water molecules in the rigid solvation layer are brought
back into the bulk phase through interactions with the added salt,
their greater freedom of movement causes a significant increase
in their entropy. Thus, ΔG becomes negative and precipitation
occurs spontaneously.
 Precipitation with organic solvents
 Addition of water miscible solvents such as ethanol or methanol
to a solution may cause proteins in the solution to precipitate.
 The solvation layer around the protein will decrease as the
organic solvent progressively displaces water from the protein
surface and binds it in hydration layers around the organic solvent
molecules.
 With smaller hydration layers, the proteins can aggregate by
attractive electrostatic and dipole forces.
 Important parameters to consider are temperature, which should
be less than 00 C to avoid denaturation, pH and protein
concentration in solution.
 Miscible organic solvents decrease the dielectric
constant of water, which in effect allows two proteins to
come close together.
 At the isoelectric point the relationship between the
dielectric constant and protein solubility is given by:
log (S) = K/D2 + log S0
 S0 is an extrapolated value of S, D is the dielectric
constant of the mixture and K is a constant that relates to
the dielectric constant of water.
 The Cohn Process for plasma protein fractionation
relies on solvent precipitation with ethanol to isolate
individual plasma proteins.
Temperature profile as a function of acetone concentration
 Polyvalent metallic ions

Metal salts can be used at low concentrations to

precipitate enzymes and nucleic acids from
solutions. Polyvalent metal ions frequently used
are Ca+, Mg2+, Mn2+ or Fe2+.
 The metal ion will bind to a part of the protein.
An advantage of this type of precipitation is that
they have great precipitating power in a dilute
solution.
 These ions can be classified into three groups.
a) Ions such as Mn2+, Fe2+, Co2+, Ni2+, Zn2+, and Cd2+
will bind strongly to carboxylic acids and to
nitrogenous compounds.
b) Ca2+, Ba+2, Mg+2, and Pb+2 will mainly bind to
carboxylic acids.
c) Finally, Ag+, Hg2+, and Pb2+ will bind strongly to
sulphydryl groups.
 Precipitation of Proteins by Non-ionic polymer
Polyethylene Glycol (PEG) or Dextran

H−(O−CH2−CH2)n−OH.

Polyethylene glycol (PEG) is a polyether compound with many applications from industrial
manufacturing to medicine. It has also been known as polyethylene oxide (PEO) or
polyoxyethylene (POE), depending on its molecular weight

 Water-soluble polymers such as dextran and PEG cause aggregation

of proteins without denaturation and can be used at ambient
temperature.

 The disadvantages of such polymers is their high viscosity.

 However, PEG 20% w/v are not too viscous and can be efficiently used
for protein precipitation.

 PEG is available commercially with various degrees of polymerization,

and the most widely used polymers are those with mwts. 6,000 and
20,000 Da.
Proteins are sterically excluded from regions of the solvent
occupied by the inert synthetic polymers and are thus concentrated
until their solubility is exceeded and precipitation occurs.

 In spite of its relatively inert chemical properties, PEG

has been found to have some interesting biological effects.

 For example, it causes profound changes in the

conformation of DNA polymers.
The attempts are made to develop its potential for the
large-scale isolation of clinically useful proteins from
human plasma.

 Because of its nondenaturing qualities, PEG provides

an attractive alternative to ethanol for recovering minor
components, many of which are difficult to obtain in
native form from ethanol-generated fractions.
  The apparent
solubilities of various
proteins (14,000 to
670,000 daltons) were
measured in the
presence of polyethylene
glycols (PEGS) of
different sizes.

 All of the solubility

curves, determined by
measuring the protein
concentration in the
supernatant of
centrifuged mixtures,
exhibited the
characteristic linear
dependence of log S
(g/liter) on PEG
concentration (%, w/v).

Figure . Effect of molecular weight of PEG on the solubility of human serum albumin.
Cohn and Ferry Equation

 Studies with purified proteins indicate that the

dependence of solubility, S, on PEG concentration, C,
is strictly exponential with data generally conforming
to the following equation (Cohn and Ferry, 1943):

log S =  C + constant

The form of this equation is identical to that used to

describe the solubility of proteins in the presence of
inorganic salts (Cohn and Ferry, 1943).
PROBLEMS
We get:
The solubility of a protein is 15 g/litre at ammonium
sulphate concentration of 2.2 M and 0.25 g/litre at 3.0 M.
Calculate the solubility of the protein at 3.8 M of the salt.

Use the Cohn eqn: log S =  - KsI

Question 1
Why do proteins exhibit minimum solubility at their pI values?
Proteins have no net charge at their pI values and the electrostatic repulsions
between protein molecules are, therefore, at a minimum at these conditions. This
results in minimum solubility of the protein at its pI.

Question 2
Why do proteins have net negative charge at high pH and net positive
charge at low pH conditions?
Answer to Question 2
The most important functional groups for charge of the protein are the amino and
carboxyl groups. At high pH there are few protons so the carboxyl groups are
ionized with a negative charge and the aminos have lost their protons and have no
charge. At low pH with abundant protons, the carboxyls are not ionized and the
aminos are protonated for a net positive charge on the protein.
Question 3
Do choatropic salts exhibit a denaturing effect on the proteins?
Answer to Question 3
Not necessarily. Chaotropic salts only disrupt water structure. They do not
necessarily denature proteins.

Question 4
Are negative ions more effective for salting out than positive ions?
Answer to Question 4
No. More negative ions are more effective than less negative ions for salting out. The
same is the case with positive ions. Solubility decreases exponentially with
increasing magnitude of charge.
Question 5
Are there any macroscopic factors that play a role in salt induced
precipitation?
Answer to Question 5
Yes. Salt-protein contacting conditions such as the type of reactor used, the rate
of mixing, the mode of addition of salt, temperature, etc. affect solubility to a large
extent
Question 6
You are given a mixture predominantly containing Lysozyme (pI = 11) and
Myoglobin (pI = 7) and are asked to precipitate Myoglobin selectively. You
have to choose between Ammonium chloride and Ammonium Sulfate as
your salt to use in precipitating. Which salt would you choose? And under
what pH conditions would you operate?
Answer to Question 6
You would choose ammonium sulfate because sulfate has greater charge than
chloride, and you would operate under neutral pH conditions because this is close
to the pI of the protein of interest
On Overview of Ammonium Sulfate Precipitation and Dialysis
We have now generated a crude extract from E. coli cells and shown that
this extract contains ß-galactosidase activity. Remember that our ultimate
goal is to get purified enzyme. So now we will begin to separate this activity
from a lot of the contaminating proteins in the crude extract. We will start this
process using a relatively crude separation method called ammonium
sulfate precipitation.

What is the theory behind ammonium sulfate precipitation as a tool to

separate proteins?
This method uses the solubility properties of individual proteins to separate them
from one another. Remember that enzymes are usually soluble proteins existing
in an aqueous environment. There are chemical groups that are charged or polar
that are shielded by solvation spheres. Because these charges are shielded,
proteins aren’t as easily able to aggregate with one another. This also helps keep
proteins in their proper and functional 3D shape.
When you add ammonium sulfate to a solution of protein, the ammonium sulfate
competes with the protein for binding to water. This effectively dehydrates the
proteins. Once the water molecules are stripped from the soluble protein, the
protein precipitates from solution.
Why is ammonium sulfate used to “salt out” proteins?
In theory, other salts could be used as well. Ammonium sulfate, however, is very
soluble, allowing for a range of concentrations to be applied. Therefore,
sequential cuts will be done: Below 25% saturation, particulate or high molecular
weight proteins precipitate. Therefore, a 25% cut may allow you to remove a lot
of “junk” from your protein prep. Generally, then experimenters will do
sequential 10 – 20% cuts above this range to see where their protein precipitates.
Above 85% saturation, little is soluble, so you can use an 85% cut to
concentrate dilute solutions of protein.
What concentration of ammonium sulfate will precipitate
out β-galactosidase?
Published papers have shown that this enzyme will precipitate out
at 33% saturation. How do we determine how much AS to add to
achieve 33% saturation?
We know that water is saturated with ammonium sulfate at 70g
AS / 100 mL water.
Therefore, a 33% saturation would be:
0.33 ( 70 g) in 100 mL = 23.1 g AS / 100 mL or 231 mg AS / mL .
How is dialysis used to clean up your protein preparation?
Your goal now it to get rid of the extra salt in the protein prep so that
you can continue with ion exchange chromatography. You will use
dialysis to do this. Dialysis depends upon the use of a dialysis
membrane with a defined pore size. Your dialysis tubing has a 10 kD
MW cut off. This means that anything with a size smaller than 10 kD
will be able to pass through the dialysis tubing. Larger molecules will
be trapped inside the tubing.
Remember that dialysis depends upon the property that molecules will
travel by diffusion towards areas of lower concentration. This process
means that ions such as AS which will start out at much higher
concentration inside the tubing will exhibit a net transfer towards the
outside of the tubing. This progress will continue until the
concentration of AS is equal INSIDE and OUTSIDE the tubing.
Because most buffer components are very small, dialysis is useful for
both desalting solutions and for buffer exchange.
What other methods exist for desalting and buffer exchange?
One method that you have already used in other courses is gel
filtration. This method of chromatography uses a resin that is made up
of beads with channels that run through each bead. This means that
small molecules can enter the beads and get held up in the column,
while larger molecules, such as protein, will stay outside of the beads
and elute first. The end result is that your protein will elute in whatever
buffer you have used to equilibrate your column.