Precipitation

important traditional method for purifying proteins and nucleic acids. Involves selective conversion of a specific dissolved component of a complex mixture to an insoluble form using appropriate physical or physicochemical means.

Factors utilized for precipitation Cooling/heating pH adjustment Addition of solvents such as acetone and ethanol Addition of anti-chaotropic salts such as ammonium sulphate and sodium sulfate (intermolecule) Addition of chaotropic salts such as urea and guanidine hydrochloride (intramolecule) Addition of biospecific reagents as in immunoprecipitation

Anti-chaotropic expose hydrophobic patches on proteins by removing the highly structured water layer which usually covers these patches in solution Chaotropic solutes increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects.

Cooling solubility of A is more sensitive to the lowering of temperature than B. Hence, the separation of A and B could be carried out by lowering the temperature of the solution to the point where A is largely precipitated while B is still largely in solution

Temperature induced precipitation Based on the assumption that that denaturation follows first order chemical kinetics with an Arrhenius dependence [p] is the dissolved protein concentrations Rate constant k is given by E can be manipulated by changes in pH or solvent

Example Two of these dehydrogenases have the precipitation rate constants You have a solution containing equal activities of each enzyme. What will be the activity be after 10 min at 20oC? What will it be after 10 min at 50oC?

Solution By integrating we see that 20oC 50oC kA 8.3 x 10-11 1.6x10-4 kB 4.0x10-10 3.0x10-3 By integrating we see that 20oC 50oC [A]/[Ao] >0.999 0.91 [B]/[B0] 0.17

Additives Precipitation by using additives is governed by the thermodynamic equation:

Additives When a solute is being precipitated from its solution, the precipitate is mainly composed of the solute. So its chemical potential at a given temperature is constant. On the other hand the chemical potential of the dissolved solute depends on its concentration and is given by:

Additives 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 of the solute in solution be the same as that in the precipitate.

Additives-Organic solvents precipitate proteins and other macromolecules by reducing the dielectric constant of the medium

Additives-Organic solvents Higher concentrations of organic solvents can denature proteins. Organic solvents usually bind to specific locations on the protein molecules and thus disrupt the hydrophobic interactions which hold the protein structure in place. very small amounts of organic solvent are used in precipitation processes and these are carried out at low temperatures to minimize denaturation

Example 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 precipitate. How much protein would precipitate if 100 ml of ethanol were added to 100 ml of a similar protein solution at the same temperature? Assume that the dielectric constant of the medium varies linearly with volumetric composition of the two solvents.

Additive-pH the minimum solubility being observed at its isoelectric point Separation of the two proteins could be carried out by maintaining the solution at isoelectric point of B or at isoelectric point of A

Addition of anti-chaotropic salts Anti-chaotropic salts such as ammonium sulphate and sodium sulphate expose hydrophobic patches on proteins by removing the highly structured water layer hydrophobic residues on a protein molecule can interact with those on another and leads to aggregation/precipitate

The constant B is the natural log of the theoretical solubility of the protein in salt free water. The Cohn equation is valid only in the salting-out region of the precipitation process. The constant B depends on the protein, the temperature and the solution pH. Ks is independent of the temperature and pH but depends on the salt and the protein.

Addition of biospecific reagents relies on antigen-antibody recognition and binding. multivalent antigens react with antibodies in solution they form large molecular networks by cross-linking which eventually precipitate (precipitins)

Protein Solubility –Rules of thumb Larger proteins are less soluble than smaller ones Protein whose surface is rich in hydrophobic amino acids are less soluble Denatured proteins are less soluble pH close to the isoelectric point decrease solubility The higher the dielectric constant the lower the solubility The higher the ionic strength the lower the solubility

Mechanism of precipitate formation precipitation is taking place as a function of time A precipitation process has the following stages: Mixing Nucleation Diffusion limited growth Convection limited growth

Mixing takes place over a finite amount of time. the amount of time depending on the properties of the components as well as on the processing conditions (mixing intensity and temperature)

Initial Mixing Initial mixing is the mixing required to achieve homogenity after the addition of a component to cause precipitation By assuming that mixing between randomly dispersed eddies is instantaneous and the mixing within eddies is diffusion limited Where ρ is liquid density, v is the kinematic viscosity and P/V is the agitator power input/unit volume of liquid

For spherical eddies of diameter le, this becomes It is necessary to mix until all molecules have diffused across all eddies. This time can be estimated from From Einstein diffusion relationship Where δ is the diffusion distance and D is the diffusion coefficients For spherical eddies of diameter le, this becomes The average eddy length (Kolmogoroff length) scale depends on volume of the system, the density and viscosity of the medium, the power input for mixing

nucleation step formation of very minute particles initiates at regions having localised supersaturation high degrees of supersaturation result in formation of gelatinous precipitates Controlled supersaturation produces amorphous precipitates which are both easy to filter and centrifuge

Diffusion limited growth leads to the formation of bigger particles the rate of particle formation is dependent on: the physicochemical properties of the protein the liquid medium The rate constant K depends on the diffusivity (D) and diameter (d) of the protein.

The constant k could be determined as Integrating gives Stokes-Einstein equation can be used to estimate the diameter of globular protein:

Example We wish to precipitate the protein α2- macroglobulin contained in a 100 liters of aqueous solution at 20oC in a tank at a concentration of 0.2 g/liter. α2-macroglobulin is a globular protein with a molecular weight of 820,000 and diffusion coefficient of 2.41 x 10-7 cm2/s at 20oC. The precipitate particles have a density of 1.3 g/cm3. The solution is stirred with 75 W motor. Calculate the concentration of nuclei at the end of the ‘initial mixing’ period

Convection limited growth The greater the extent of mixing, the greater is the frequency of collision. The rate of precipitate formation of a particular size in convection limited growth is given by: The rate constant φ depends on: the size and sticking tendency of the particles, the volume of the system, the density and viscosity of the medium the power input for mixing.