Precipitation with Organic Solvents

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 ---78.5 Methanol ---32.6 Ethanol ---24.3

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 0 20 40 60 80 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. calmodulin % Native structure Average protein 0 100 Temp, C Calmodulin is an excellent example of a protein that can be purified by thermal denaturation. Thermal Denaturation. • Proteins differ in their thermal stability and ability to renature after thermal denaturation. • 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. 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 4850 170 28.50 97 4.07 Acetone 4600 119 38.65 92 5.25 Ethanol 3950 98 40.30 79 5.75 Isopropanol 4200 106 39.62 84 5.66 Acetic acid 3600 90 40.00 72 5.71 PEG 4000 4200 106 39.62 84 5.66 PEG 6000 3800 106 35.84 76 5.12 PEG 20000 3500 108 32.40 70 4.60 An overview 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