1. Precipitation with Organic Solvents
Polar organic solvents such as aliphatic alcohols (ethanol) and ketones
(acetone) reduce protein solubility.
Decreasing surface hydration and increasing hydrophobic surface
promoting aggregation like salt effect.
However, such effects are less involved because of solubilizing
influence of organic solvent on these areas.
The principal effect is the reduction in water activity. There is medium
decrease in the dielectric constant with the addition of an organic solvent
leading to the decrease in the solvating power of water for the charged,
hydrophillic protein molecule, and thus protein solubility decreases and
precipitation occurs. This is described by the equation:
log S = A/e2 + log S0
S is solubility in presence of a solvent and S0 is the original solubility. A is
the constant depending on temperature and protein employed and e is
dilectric constant depending on the type of solvent used.

2. Precipitation With Organic Solvents
- Reduction of dielectric constant of the solvent
* Dielectric constant is a measurement indicating the easiness to
separate two differently charged (+, -) molecules. The lower the
dielectric constant of the solvent, the easier the two differently
charged molecules contact with each other.
H2O
Methanol
Ethanol

3. Precipitation Organic solvent precipitation
Volume of miscible organic solvent to be added to 1 liter of solution
At C1% vol/vol liquid, to take it to C2% vol/vol
Volume (ml) = 10(C2 – C1)/100 – C2

4. Precipitation with Organic Solvents
Protein denaturation is the major drawback to this approach and is
largely circumvented by using polyalcohols.
Efficient precipitation method for cytoplasmic and other water
soluble proteins, less efficient for hydrophobic membrane proteins

5. Precipitation with Organic Solvents
The principal causes of aggregation are likely to be electrostatic and
dipolar van der Waals forces

6. Precipitation with Organic Solvents
The size of protein molecule is an important factor for aggrgation;
the larger the molecule, the lower the percentage of organic solvent
required to precipitate it
6.0
phosphorylase
pyruvate kinase
lactate dehydrogenase
5.0
enolase
Log molecular weight
phosphoglycerate kinase
myokinase
parvalbumin
4.0
Acetone concentration (% vol/vol)

7. Organic solvent precipitation: practical considerations
Most enzymes precipitate with acetone in the range of 20 – 50% vol/vol.
Exact percentages are difficult to define.
Temperature must be kept very low around 0 ˚C to avoid denaturation
Addition of solvent to water causes heat evaluation due to hydration of solvent
molecules. Consequently slow addition with efficient cooling should be followed
Protein concentration should be around 5 – 30 mg/ ml,
Salt concentration should not be high, otherwise electrostatic aggregation
is impaired. Optimum concentrations are 0.05 – 0.2 M
Don´t leave the protein precipitate in solvent for long
Less than 10% vol/vol solvent concentration doesn´t effect the further purification
processes except affinity and hydrophobic interactions

8. Precipitation with Organic Solvents

9. Organic solvent precipitation: practical considerations
Denaturation at elevated temperatures
Intramolecular hydrophobic interactions help maintain protein structure
and stability
At low temperatures, there is lack of conformational felxibility, which means
organic solvent molecules don´t get access to penetrate the internal structure
and cause destablization
At above about +10 ˚C denaturation effects become subtantial, since small
organic molecules enter through the cracks caused by flexing of the structure
They attach themselves to hydrophobic residues and thus destabilize the
intra-hydrophobic forces, which result in autocatalytic denaturation

10. Selective denaturation by organic solvent

11. Isoelectric (pI) precipitation
Precipitation is caused by changing the pH of the protein solution.
This effect is due to the different ionic groups of a protein molecule.
At isoelectric point (pI) where the net charge on a protein is zero,
the electrostatic repulsions between molecules are at a minimum and
result in aggregation due to predominating hydrophobic interactions.
Several proteins have very close pIs, and in a protein mixture different
proteins with similar properties coprecipitate and aggregate. Thus, most
isoelectric precipitates are aggregates of many different proteins.

12. Isoelectric (pI) precipitation: practical considerations
Since precipitation is carried out away from physiological pH, it is
important to make sure protein is stable at that pH.
Isoelectric precipitation is more useful if combined with other modes
of precipitation, e.g., organic solvent or PEG addition.
Choice of acid or base for pH adjustment will depend on individual
protein system.
While milder acids or bases are mainly used to bring change in pH, in
some systems abrupt changes are neccessarily achieved by using strong
acids or bases.
The way of addition is important. Rapid precipitation can be in some
systems useful for producing larger aggregates.

13. Heat/pH-induced precipitation
The precipitation strategy involve the option to use heat or pH to denature
and precipitate the unwanted proteins while the desired protein remains
unaffected
Known as subtractive adjunct precipitation
Some proteins such as adenylate kinase, plant protein inhibitors, trypsin and
certain proteins of thermophillic organisms are relatively heat stable
Similarly some proteins are biologically active outside normal pH range of
5 – 10.
This approach is now commonly used for single-step purification of thermo-
stable enzymes expressed in mesophilic host

14. Thermal Denaturation.
Proteins differ in their thermal stability and ability to renature after thermal denaturation.
Calmodulin is an excellent example of a protein that can be purified by thermal denaturation.
Average
protein
calmodulin
% Native structure
Temp, C
100
In general, smaller, highly charged proteins are stable to higher temperatures than large, more hydrophobic proteins.
A major limitation to the use of this technique is the action of proteases which are inherently resistant to denaturation

15. Precipitation with polymers
Nonionic water soluble polymers
Several nonionic water soluble polymers cause precipitation of proteins
however, high viscosity of many of them make their use rather difficult
Polyethylene glycol (PEG), one exception which can be used thoroughly
because upto 20% (w/v) of this solution is not viscous. Available in variety
of degrees of polymerization
H-(CH2-CH2-O)n-H polymer of ethylene oxide
High molecular weight called polyethylene oxide (PEO) and low molecular
weight called polyethylene glycol (PEG)

16. Precipitation with polymers
Nonionic water soluble polymers
PEG precipitation is somewhat similar to organic solvent precipitation, and
PEG molecules are regarded as polymerized organic solvent
Solubilities of protein decrease exponentially with increasing concentration
of polymer according to:
log S = log S0 – βC
S and S0 solubility in presence and in absence of PEG, respectively and C is
concentration

17. Precipitation with polymers: practical considerations
Nonionic water soluble polymers
PEG precipitation is quite selective for fractionating plasma/serum proteins,
so has found wide application in clinical diagnostic testing e.g., prolactin,
protein S measurements
PEG with an average molecular weight of 4000 – 6000 is commonly used
Very mild and selective method, less tendency to denature proteins
Disadvantge is that PEG is not easily removed from the protein fraction.
However, polymer can be removed in subsequent stages of purification scheme

18. Precipitation with polymers: practical considerations
PEG Precipitations – some examples
Thyroid stimulating immunoglobulins
Fractionating Collagen types
Acid phosphatases
Lactoglobulins
Lipases
Nucleic acid precipitations and purifications

19. An overview
Precipitation of lipase by different precipitating methods from
cell culture filtrate
Specific
Total Total activity Recovery of
Precipitating activity protein (U/mg enzyme Fold
agent in ppt (U) ppt (mg) protein) (%) purification
Ammonium sulfate
Acetone
Ethanol
Isopropanol
Acetic acid
PEG
PEG
PEG
Initial total activity of the enzyme = 5000 U; protein = 710 mg

20. Precipitation with polymers: practical considerations
PEG Precipitations
Advantages
Precipitation with polymers retains more intact structure of the proteins
as compared to other precipitation modes – results in higher bioactivity
of the protein
Bottleneck
Removal of polymer after precipitating the protein –
Low concentrations of the polymer are removed in subsequent procedures
Ultrfiltration or salt induced phase separation can be used

21. Precipitation with polymers
Synthetic and Natural Polyelectrolytes
Homopolymer
Copolymer
Random copolymer
Block copolymer
Branched polymers, dendramers

22. Precipitation with polymers
Synthetic and Natural Polyelectrolytes
Homopolymer
-A-A-A-A-A- or -B-B-B-B-
Random copolymer
-A-B-B-A-B-B-A-A---
Block copolymers
-A-A-A-A-A-A-B-B-B-B-B-B-
Branched polymer, dendramers

23. Precipitation with polymers
Synthetic and Natural Polyelectrolytes
Polyanions and polycations interact with proteins below or above the
isoelectric points
These interactions may result in soluble complexes or formation of
amorphous precipitates
This may be achieved by the selection of the polyelectrolyte, choice
of the ionic strength and pH
Protein precipitation by polyelectrolytes may lead to closely packed
aggregates that are conveniently separated by settling or can generate
open textured aggregates that can be separated by filtration
The precipitated proteins are recovered from the insoluble protein-
polyelectrolyte complex aggregates by redissolution achieved by pH
or ionic strength adjustment

24. Precipitation with polymers
Polyelectrolyte complexes + - + -
- -
Polycation &
Polyanion a) + + + -
Polycation &
protein + + + + - + + b) - + - + - + - + + - - + + - - + + - - + + - - - -
Polyampholyte &
protein - + - c) + + + + + - +

25. Precipitation with polymers
Synthetic and Natural Polyelectrolytes
Use of polyelectrolytes as precipitating agent offers several advantages even
though their costs may be high
Generally, very low concentrations of the polyelectrolytes are required,
can also be recycled, fractionation potential appears promising
Both pH and ionic strength are the important determinent in the
precipitation efficiency. Increasing ionic strength reduces protein-poly
electrolyte interactions, thus evidencing complex formation through
electrostatic interactions.
Polyethylene imine (PEI), a polycation and polyacrylic acid (PAA), a
polyanion are generally used

26. Precipitation with polymers
Synthetic and Natural Polyelectrolytes
Polyelectrolyte precipitations have inherent selectivity with regard to
differently charged species and change of medium conditions also exerts
such selectivity
Example: Lysozyme and Hemoglobulin are both precipitated by polyacrylic
acid (PAA).
Lysozyme precipitating quantitatively in the pH range of 4.5 – 6.5
Hemoglobulin precipiting quantitativley in the pH range of 4.25 – 5.25
(Lysozyme having more basic character)

27. Precipitation with polymers
SPECIFIC: AFFINITY PRECIPITATION
Homobifunctional mode
Lattice formation
Immunoprecipitation, agglutination
Divalent antibody – multivalent antigen reaction
Introduced by Larson and Mosbach (1979)
Lactate dehydrogenase (tetrameric)
Glutamate dehydrogenase (hexamer)
Avidin (tetramer)
ligands
bis-NAD, bis- derivatives of triazine dyes, iminobiotin