Precipitation, Bioseparation

Precipitation

• Protein precipitation achieves separation by the conversion of soluble proteins to an insoluble state, which subsequently can be removed by various means.

• Precipitation results in both concentration and purification.• Often used early in the sequence of downstream purification,

reducing the volume and increasing the purity of the protein prior to any chromatography steps.

• A protein is made insoluble by changing its surface charge characteristics or changing the solvent characteristics; (the latter being preferred).

• The greater the initial concentration of the desired protein, the greater the efficiency of precipitation.

• A typical globular protein in aqueous solution exhibits a non-uniform distribution of surface positive & negative charges.

• The surface also exhibits hydrophilic & hydrophobic regions.• Solubility of the protein is determined by the interaction

between the surface regions with surrounding water molecules.

• Solubility is the property of a substance (solute) that determines the extent to which it dissolves in a liquid (called the solvent) and form a homogenous solution.

• It is based on factors: – temperature, – compatibility between the solute’s net charge and the solvent’s

polarity, – the formation of hydration layers,– the presence and concentration of other solutes dissolved in the

solvent, and – solvent’s ionic strength and dielectric constant.

• Macromolecules (such as proteins and nucleic acids) are typically present in aqueous solvents.

• They are able to dissolve as a result of assuming a stable conformation in the solvent and then being surrounded by hydration layer(s).

• When placed in an aqueous solution, the macromolecules adopt a structure in which – most hydrophobic (nonpolar) portions of the molecules gather

inwards, and – most hydrophilic (charged/polar) portions surround the exterior,– guided to a final conformation that has the lowest Gibbs free energy.

• The attraction between the hydrophilic surface of the macromolecule and the polar water molecules creates an interface in which similarly-oriented water molecules associate with, and surround, the solute’s surface to form a highly ordered layer, called the hydration layer (also called the interfacial double layer).

• The formation of hydration layers enhances the solubility of macromolecules by greatly reducing inter-macromolecular dipole-dipole attraction and thus preventing their association with other solutes.

• Proteins in their natural condition generally exhibit a net negative charge on the surface and attract positive ions to form a layer of counter ions close to the protein surface - Stern layer

• This layer is surrounded by a diffuse layer of mobile counter ions – Guoy-Chapman layer

• The stability of the protein – electrolyte colloid is due to the balance between attractive & repulsive forces between colloidal particles not allowing them to form aggregates.

• The stern layer: controls the effective thickness of the outer layer and thus determines the stability of the colloid.

• The thickness of these two layers can be reduced by– changing the solvent characteristics: ionic strength & dielectric

constant or– by changing the protein surface characteristics

• Thereby bringing about a decrease in the solubility of a protein.

Methods for precipitation

• Broadly divided into two groups:• Where protein solubility is reduced and precipitation is

brought about by altering some physico-chemical property of the solvent (pH, dielectric constant, ionic strength & water availability

• Where precipitation is induced by a direct interaction between the protein and a precipitating agent.

Based on

Solvent property modification• pH change (Isoelectric

precipitation)• Ionic strength change

(Salting out)• Change in dielectric

constant (Organic solvent mediated)

• Change in water availability (by non-ionic polymers)

Solute property modification• Selective interaction with

metals, polyelectrolytes or affinity reagents to precipitate protein of interest.

• Selective denaturation to precipitate unwanted proteins– pH denaturation– Thermal denaturation– Organic solvent denaturation

Advantages of precipitation steps in downstream processing

1. Reduction in the volume of fermentation broth by a factor of 10-50 times

2. Concentration of the desired product3. Rapid separation and stabilization esp of labile products4. Convenience to hold the complex and the lengthy

downstream processing at an intermediate stage and diversify into different process steps to achieve product isolation and purification

5. A less expensive and robust methodology at industrial scale operations to achieve desired degree of purification.

Isoelectric precipitation

• The solubility of protein depends on, among other things, the pH of the solution.

• Similar to the amino acids that comprise protein, protein itself can be either positively or negatively charged overall due to the terminal amine -NH2 and carboxyl (-COOH) groups and the groups on the side chain.

• It is positively charged at low pH and negatively charged at high pH.

• The intermediate pH at which a protein molecule has a net charge of zero is called the isoelectric point of that protein.

• The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein becomes zero.

• At a solution pH that is above the pI the surface of the protein is predominantly negatively charged and therefore like-charged molecules will exhibit repulsive forces.

• Likewise, at a solution pH that is below the pI, the surface of the protein is predominantly positively charged and repulsion between proteins occurs.

• However, at the pI the negative and positive charges cancel, repulsive electrostatic forces are reduced and the attraction forces predominate.

• The attraction forces will cause aggregation and precipitation.

• The pI of most proteins is in the pH range of 4-6. • Mineral acids, such as hydrochloric and sulfuric acid are used

as precipitants.• The greatest disadvantage to isoelectric point precipitation is

the irreversible denaturation caused by the mineral acids.• For this reason isoelectric point precipitation is most often

used to precipitate contaminant proteins, rather than the target protein.

• Eg. Casein precipitation during cheese making. • When microorganisms grow in milk, they often produce acids

and lowers the pH of the milk.

• For casein, the IEP is approximately 4.6 and it is the pH value at which acid casein is precipitated.

• In milk, which has a pH of about 6.6, the casein micelles have a net negative charge and are quite stable.

• During the addition of acid to milk, the negative charges on the outer surface of the micelle are neutralized (the phosphate groups are protonated), and the neutral protein precipitates.

• The same principle applies when milk is fermented to curd.• The lactic acid bacillus produces lactic acid as the major

metabolic end-product of carbohydrate [lactose in milk] fermentation.

• The lactic acid production lowers the the pH of milk to the IEP of casein. At this pH, casein precipitates.

Effect of pH changes in altering the solubility of the protein

Isoelectric preciptation

• The relationship between the solubility of a protein and increasing ionic strength of the solution can be represented by Cohn equation:

• S = solubility of the protein, B is idealized solubility, K is a salt-specific constant and I is the ionic strength of the solution, which is attributed to the added salt.

• zi is the charge of the ion and ci is the concentration of the ion.

Precipitation by addition of salts

• Most enzymes exists in cell fluids as soluble proteins even at high concentrations (upto 40%)

• This is due to combined electrostatic effects consists of:– Polar interactions with aqueous solvent– Ionoic interactions with salts present– Repulsive electrostatic forces between protein molecules &

aggregates of like charges

• The increase in protein solubility with increasing salt concentration in the ionic strength range of 0 to 0.5M at a given pH and temperature is called salting in.

• The solubility of a protein at low ion concentrations increases as salt is added, a phenomenon called "salting in".

• The additional ions shield the protein's multiple ionic charges, thereby weakening the attractive forces between individual protein molecules (such forces can lead to aggregation and precipitation).

• However, as more salt is added, the solubility of protein again decreases.

• This is called "salting out" effect.• It is primarily a result of the competition between the added salt

ions and the other dissolved solutes (protein molecules) for molecules of solvent (water).

• At very high salt concentrations, so many of the added ions are solvated that there is significantly less bulk solvent available to dissolve other substances, including proteins.

• Since proteins precipitate at different salt concentrations, salting out is the basis of one of most commonly used protein purification procedures.

• Adjusting the salt concentration in a solution containing a mixture of proteins to just below the precipitation point of the protein to be purified eliminates many unwanted proteins from the solution.

• Then, after removing the precipitated proteins by filtration or centrifugation, the salt concentration of the remaining solution is increased to precipitate the desired protein.

• The precipitation of desired protein is then dissolved in water to make a solution of this protein.

PRECIPITATION - II

Precipitation with Ammonium Sulfate

• Inorganic salts can be utilized for the precipitation of proteins, with ammonium sulfate being the most common.

• The advantages of ammonium sulfate are:– at saturation, it is of sufficiently high molarity that it causes the

precipitation of most proteins; – it does not have a large heat of solution, allowing heat generated to

be easily dissipated; – its saturated solution (4.04 M at 20 C) has a density (1.235g cm-3) that

does not interfere with the sedimentation of most precipitated proteins by centrifugation;

– its concentrated solutions are generally bacteriostatic; and – in solution it protects most proteins from denaturation.

• Effects of Solute Net Charge• In an aqueous solvent solutes with a higher net charge are

more soluble, due to their enhanced ability to associate with water molecules and create a hydration layer, and their lessened inter-solute interactions as a result of increased repulsion from like-charges.

• Effects of Solvent Ionic Strength• Increasing a solvent’s ionic strength (the concentration of ions

in solution) decreases the availability of unassociated water molecules, which are required for the formation of hydration layers. This increases intermolecular attraction between solutes and promotes coalescence, thus decreasing the solute’s solubility.[

• Isoelectric Precipitation• At its isoelectric pH, a macromolecule possesses no net

charge and thus no like-charge repulsion occurs between solutes.

• This results in weaker interactions with water molecules and makes the macromolecule more hydrophobic, increasing its affinity for combining with other solutes, and thus allowing for precipitation.

• Isoelectric precipitation is the process by which the pH of the solution is manipulated to reach the solute's pI to achieve this effect.

• A common method of protein precipitation is called salting out, and is achieved by increasing an aqueous solvent’s ionic strength by dissolving a salt.

• Increases in salt concentrations steadily decrease the solubility of proteins in the solution, until the proteins become insoluble and precipitate out of solution.

• Since proteins generally differ in their solubility, slowly adding salt allows for selective precipitation of dissolved proteins in order of lowest to highest solubility.

• The most commonly used salt, (NH4)2SO4 due to its high solubility, lack of buffering capacity, minimal cost, and the low density of the resulting solution relative to other salts, which aids in centrifugation separation.

Dialysis

• Semi-permeable membrane is containing pores of less than macromolecular dimensions.

• These pores allow small molecules, such as those of solvents, salts, and small metabolites, to diffuse across the membrane but block the passage of larger molecules.

• Cellophane (cellulose acetate) is the most commonly used dialysis material although many other substances such as nitrocellulose and collodion are similarity employed.

• So, dialysis is a method in which an aqueous solution containing both macromolecules and very small molecules which are placed in a dialysis bag which is in turn placed in a large container of a given buffer or distilled water.

• Thus small solute molecules freely pass through the membrane, and after several hours of stirring the equilibrium will reach (the concentration inside and outside the bag are the same).

• Thus, at equilibrium the concentration of small molecules outside and inside the bag is the same while the macromolecules remain inside the bag.

• During dialysis the external fluid should be changed in order to reach the required composition inside the dialysis bag.

• There are three factors that affecting the rate of dialysis: the first is the concentration differences of that molecules between the internal and external solution (which is the driving force for the movement of the molecules).

• The second is mixing on both sides of dialysis membrane will increase the rate of movement prevent the small particles on the side of low concentration.

• The third is dialyzable particles size versus pore size of the membrane, substances that are very much smaller than the pore size will reach equilibrium faster than substances that are only slightly smaller than the pores.

• The main point to be noted is that there is a rapid initial drop in dialysis process followed by a slow approach to equilibrium.