1. Precipitation

2. Introduction
Important traditional method of purification of proteins and nucleic acids
Selective conversion of a specific dissolved component of a complex mixture into an insoluble form by
Physical factors
Physicochemical factors

3. Factors utilized for precipitation
Cooling
pH adjustment
Addition of solvents such as acetone and ethanol
Addition of anti-chaotropic salts such as ammonium sulphate or sodium sulphate
Addition of chaotropic salts such as urea and guanidine HCl
Addition of biospecific agents (affinity precipitation): e.g immunoprecipitation)

4. Temperature dependence of protein solubility
Solubility of proteins in aqueous solutions is dependent
on temperature
Protein B
Solubility
Cooling is an integral part of solvent and salting out but rarely used alone
Protein A
Temperature

5. Precipitation heuristics
Precipitation at low temperature increases yield and reduces denaturation
Optimum ionic strength for precipitation is 0.05 – 0.2 M
Higher ionic strength require excess solvent
More dilute solutions yield a finely divided which is difficult to filter
High molecular weight solutes require less solvent to initiate precipitation (Scopes, 1982)
The solubility of one protein is usually decreased in the presence of other proteins
If precipitated protein does not redissolve, it is denatured
Denaturation compromises yield

6. Thermodynamic theory of precipitation using additives
At equilibrium, the chemical potential of the solid precipitate is the same as
that in solution
The standard reference state chemical potential can be increased by adding
substances such as salts and solvents
In the presence of such additives the solute concentration in the solution phase must decrease in order that the chemical potential is equal to that in the precipitate

7. pH dependence of protein solubility
pH adjustment is rarely used alone
pH solubility effect can be utilized in salt based precipitation
Protein A
Solubility
Protein B
pIA
pIB pH

8. Effect of anti-chaotropic agents
Protein A
Solubility
Salting in
Protein B
Salting out
Ammonium sulphate concentration

9. Effect of chaotropic agents
Precipitation by denaturation
by disrupting intra-molecular hydrogen bond and hydrophobic interactions
Not used in protein fractionation
but for protein refolding (inclusion body processing)
Useful in DNA and RNA purification as they precipitate only proteins

10. Immunoprecipitation Relies on antigen-antibody recognition and binding
Isolates a certain antigen from the multitude of antigens in a solution.
When multivalent antigens react with antibodies they form large molecular networks by cross-linking
These complexes called precipitins can be removed from solutions by solid-liquid separation techniques
Another way of immunoprecipitation is by treating an antigen with insoluble antibody

12. Examples of ligands and receptors

13. Precipitation using organic solvents
Organic solvents precipitate proteins by reducing the dielectric constant of the medium
Higher concentration of solvents denature proteins by disrupting the hydrophobic interactions
To minimise denaturation small concentrations of solvents are at low temperatures
Acetone and aliphatic alcohols such methanol, ethanol, propanol and butanol are commonly used for protein precipitation
Extent of denaturation increases with increased chain length
The governing equation is
DNA and RNA can also be
precipitated using ethanol at -20oC
Cohn fractionation method for
plasma proteins
Dielectric constant for water 78.3
Dielectric constant for ethanol 24.3

14. Problem The solubility of ovalbumin in water is 390 kg/m3
When 30 ml of ethanol was added to 100 ml of a 50 mg/ml ovalbumin solution in water 33% of the protein was found to be precipitated.
How much protein would precipitate if 100 ml of ethanol were added to 100 ml of a similar protein solution at the same temperature?
The dielectric constant of pure ethanol is 24.3 and that of pure water is 78.3
Assume that the dielectric constant of the medium varies linearly with volumetric composition of the two solvents

15. Solution
When 33% of the ovalbumin is precipitated the concentration present in the solution is 33.5 mg/ml.
The dielectric constant of the solution obtained by mixing 100 ml of ethanol
with 100 ml aqueous solution is 51.3
S = mg/ml
Therefore the percentage of protein precipitated is 99.6%

16. Effect of anti-chaotropic agents
Protein A
Solubility
Salting in
Protein B
Salting out
Ammonium sulphate concentration

17. Salting out of proteins
COO- NH4+
SO42-
When large amounts of salt are
added to the solution the
ionized groups may form less
soluble ion pairs leading to
precipitation
NH4+
NH3+ SO42-
NH3+

18. The Hofmeister series of anions and cations
The arrows indicate increasing salting-out effect and chaotropic effect
Anions: Increasing “salting-out” effect
Cations: Increasing chaotropic effect
Salts promoting the molal surface tension of water

19. Precipitation using anti-chaotropic agents
Antichaotropic or kosmotropic salts
(NH4)2SO4 and NaCl
Salting in effect at lower concentrations
The increase in the solubility of proteins can be attributed to these salts providing a distinct double-layer around the protein molecules
Presence of the double layer provides the required stability to keep the protein molecules in solution and hence increases their solubility
Salting out at higher salt concentrations
Electrostatic double layer becomes diffuse
Reduced protein-protein electrostatic repulsion forces leading to aggregation

20. Precipitation using anti-chaotropic agents
The Cohn equation describes
the precipitation of proteins by salts
B = constant
Ks = salting out constant
Cs = salt concentration
Salt induced precipitations usually are carried out at low temperatures (4oC)
Synergistic effect at low temperatures
Enhanced protein stability at lower temperatures
Constant B is the natural log of the theoretical solubility of the protein in salt free water
Constant B depends on
a) protein, b) temperature, and
c) the solution pH
Ks is independent of the temperature and pH but depends on the salt and protein

21. Precipitation using anti-chaotropic agents: Operational approaches
Direct addition of solid ammonium sulphate crystals to the sample
Preferred in small scale applications
Suffers from uneven precipitation due to to localized zones of higher and lower concentration
Addition of a saturated ammonium sulphate solution to the sample
Most preferred method
Ammonium sulphate cut
Use of (NH4)2SO4 concentration in the precipitating medium which corresponds to 50% saturation of the solution
30-50% (NH4)2SO4 cut means precipitation using 30% (NH4)2SO4 followed by precipitation of the supernatant with 50% (NH4)2SO4

22. Problem
Ammonium sulphate is used to precipitate humanized monoclonal antibody from 10 litres of cell culture media, the initial concentration of the antibody in this liquid being 0.5 mg/ml.
Solid ammonium sulfate is added to the liquid such that the concentration of the salt is 1.5 kg-moles/m3. This results in the precipitation of 90% of the antibody.
When the ammonium sulphate concentration of the mixture I raised to 1.75 kg-mole/m3 a further 76.5% of the remaining antibody is precipitated.
Predict the ammonium sulphate concentration needed for total antibody precipitation. What is the solubility of antibody in ammonium sulphate free aqueous medium?

23. Solution
The antibody concentration in solution after 1.5 M ammonium sulphate
precipitation is 0.05 mg/ml = 3.2 x 10-7 kg-mole/m3
The antibody concentration in solution after 1.75 M ammonium sulphate precipitation is mg/ml = kg-mole/m3
Using Cohn’s equation
Solving the two equations we get
B = -6.26
KS = 5.8 m3/kg-mole
For total precipitation we have to assume the value of S as low as possible but not zero.
Assuming a value of S = 6 x kg-mole/m3, we get CS = 2.97 kg-mole/m3 = 2.97 M
B = ln(solubility). Therefore, the antibody solubility is = 1.91 x 10-3 kg-mole/m3
= 296 kg/m3

24. Mechanism of precipitate formation
Time dependent process
Four stages
Mixing
Nucleation
Diffusion limited growth
Convection limited growth
Methods to measure extent of precipitation
Nephelometry
Spectrophotometry
Turbidity
Time

25. Stage I Initial Mixing Mixing is not an instantaneous process
Time required for mixing to homogeneity is given by
t = mixing time (s)
l = average length of eddy (m)
D = diffusivity (m2/s)
The size of the eddies can be estimated from the equation
ρ = solution density
ν = kinematic viscosity
P/V = power input per unit volume

26. Stage II Nucleation Involves formation of very minute particles
The particles initially formed are multimers i.e., aggregates of three or four protein molecules
Nucleation usually initiates at regions having localised supersaturation due to uneven mixing
The extent of supersaturation determines nature of the precipitate
very high degrees of supersaturation result in formation of gelatinous precipitate which are difficult to filter
controlled supersaturation produces amorphous precipitates which are easily separated by filtration and centrifugation

27. Stage III Diffusion limited growth
Deposition of more proteins on small nucleation particles takes place by diffusion controlled mechanism and is a second order process
Rate constant k is dependent upon diffusion coefficient
Dependent on physicochemical properties of the protein and the medium
yi = concentration of the solute particles
k is the rate constant of the process
D is inversely proportional
to the particle diameter;
the product Dd is constant
and hence k
N = Avagadro number
D = diffusion coefficient
d = particle diameter
As determination of yi is difficult the equation can be modified to include average molecular weight

28. Stage IV Convection limited growth
The particles produced by diffusion of more proteins further increase in size by
collision with one another (absorbing momentum)
Growth in this region follows second order kinetics
The rate constant is given by
“Sticking constant” (α) is an empirical factor inserted to account for the fact that all the collisions do not produce growth
Square of the average velocity gradient
caused by the mixing
Direct integration of the equation is complicated by the fact that the particle diameter and hence the rate constant k are function of time
We can dodge this complication by considering volume fraction of solute

29. The volume fraction
The rate equation and rate constant in terms of φ
Upon integration we get
The change in particle concentration in the flow dominated regime
follows first order kinetics and shows an exponential decay

30. Flocculation period