Biochemical Techniques 06 Salt Fractionation of Proteins. Biochemistry

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2 Description of Module Subject Name Paper Name 12 Module Name/Title 2

3 1. Objectives Understanding the concept of protein fractionation Understanding protein fractionation with salt 2. Concept Map 3. Description 3.1 Protein purification Protein purification is vital for the characterization of protein structure, function and interactions, for example conformational alterations, substrate specificities, specific activities as well as interaction with other ligands. Further, for applications in the food and pharmaceutical industry, a high level of protein purity is 3

4 essential and the desired protein must be purified over a number of steps. This is thus achieved through methods of protein purification. Purification is a multistep procedure. The various steps in the purification process are aimed removing nonprotein components and impurities to finally separating out the desired protein in its pure form. 3.2 Fractionation of proteins The first step in purifying intracellular proteins is preparing a crude extract. The extract will contain a complex mixture of proteins from cell cytoplasm, and additional components such as macromolecules, cofactors and nutrients. The debris are removed by centrifugation and supernatant (crude protein extract) recovered. Crude preparations of extracellular proteins may be obtained by removing cells by centrifugation. The extract can be subjected to treatments that separate proteins into different fractions based on several properties such as size, charge etc. This process is known as fractionation. Fractionation helps in the removal of any other contaminating material and also in the enrichment of the desired protein fraction. Early fractionation steps utilize the difference in protein solubility. The solubility of a protein depends on the concentration of dissolved salts, polarity of the solvent, ph, temperature. Some or all of these variables can be manipulated to precipitate specific proteins from the solutions while others remain soluble. 3.3 Fractionation of proteins by precipitation with salt A common step to purify a protein from a crude extract is by precipitation in a solution with high osmotic strength (i.e. salt solutions). Protein precipitation is usually done using ammonium sulfate as the salt. Different proteins precipitate in different ammonium sulphate concentrations, thus separating the overall protein into several fractions. High molecular weight proteins precipitate in lower ammonium sulfate concentrations. Salt fractionation of protein does not usually lead to a highly purified protein. As mentioned above, it helps in elimination of unwanted proteins and in further concentrating the sample. Salts in the solution are then required to be removed by dialysis or gel exclusion chromatography Salting out with ammonium sulphate Salting out is an effective means for purification which explores the reduced solubility of proteins present in a solution of very high ionic strength causing certain proteins to precipitate. Figure 1 below shows salting in and salting out processes 4

5 Figure 1 Salting in and salting out Each protein molecule in solution is uniformly layered by an essential layer of hydration by water molecules which enable the molecule to repel each other and stay in solution. As more and more salt is added to the protein, the solubility of the salt added gradually tends to become higher than protein. Owing to the increased affinity of salt molecules for water over protein molecules, the hydration shell around the protein molecule is thus gradually displaced by the increasing ionic concentration in the solvent. In other words, the protein molecules are thus stripped off their hydration layer, allowing hydrophobic interaction between proteins (interactions between hydrophobic patches on protein surface) to predominate which leads to aggregation of the protein molecules and precipitation. It is important to note that salting out occurs at high salt concentration. Use of salt at very high concentrations also cause a further increase in surface tension, inducing the protein to aggregate, resulting in salt-precipitation. 5

6 Figure 2. Effects of salt on protein precipitation How is fractionation achieved Ammonium sulphate is convenient, widely used and effective chemical because it is highly soluble, cheap, less toxics and stabilizes most proteins/enzymes How does fractionation take place Fractionation of protein mixtures by the stepwise increase in the ionic strength of the salt being used for protein precipitation can prove to be an effective strategy of obtaining partially purified enzymes. For example, the salt concentration in a solution containing several proteins can be adjusted to just below the precipitation point of the protein (to be purified). This eliminates many unwanted proteins. Precipitated proteins may be removed by filtration or centrifugation, following which the salt concentration is increased further in the remaining supernatant solution to precipitate desired protein. Once the desired protein is precipitated along with many other proteins, which precipitated in presence of similar ionic strength, the precipitate is removed to obtain a fairly more concentrated fraction that now contains the desired protein. Salt concentration may be further increased in the supernatant again to precipitate remaining proteins. This is how salt fractionation of proteins takes place. At very high salt concentrations, i.e., when the protein solution is saturated with salt, all proteins may precipitate all together completely (Figure 3). 6

7 Figure 3. Steps in salt fractionation The stepwise precipitations of proteins by addition of increasing amounts of ammonium sulphate to the crude extract, with intermittent centrifugation steps is known as ammonium sulphate cuts. Amounts of solid ammonium sulphate which is to be added to a given volume of protein extract to achieve desired percentage saturation can be looked up in tables. Ammonium sulphate in solid powdered form, should be added slowly, in small batches, with continuous stirring, at low temperature, to allow for a uniform increase in concentration and ensure rapid equilibration. After all the ammonium sulphate that has been weighed out has been added and solubilized, the salt containing protein mixture is allowed to stand for sometime to allow for precipitated proteins to settle down which can be removed by centrifugation. 3.4 The Hofmeister Series The effectiveness of the different ions towards protein precipitation was established by Franz Hofmeister in 1888 and the ordering of cations and anions arranged in order of their effectiveness are called Hofmeister series. Cations: N(CH 3) 3 + > NH 4 + > K + > Li + > Mg 2+ > Ca 2+ > Al 3+ > guanidinium Anions: SO 4 2- > HPO 4 2- > CH 3COO - > citrate > tartrate > F - > Cl - > Br - > I - > NO 3 - > ClO 4 - > SCN - 7

8 Between cations and anions, the anions have the greatest effect on protein precipitation. The starting molecules decreasing solubility of the non-polar molecules and strengthen hydrophobic interactions, thus salting out the system. Contrarily, the latter molecules tend to form strong ionic interactions with the protein that disrupt hydrogen bonding, thus contributing to the denaturation of the protein. Increase in surface tension of water by salt follows the Hofmeister series. Salts which favour salting out raise the surface tension of the water the highest. As ammonium sulphate has a high solubility than any other phosphate salts, it is most commonly chosen reagent for salting out. 3.5 Dialysis After precipitating the protein fraction (using above approaches) and re-dissolving it in buffer, it is essential to remove the ammonium sulphate from the protein sample before subjecting it to subsequent steps during purification. One of the most widely used methods to achieve this is to dialyse the solution. Dialysis leads to the separation of protein molecules from other small molecules, such as salt, by using a semipermeable membrane (for e.g., cellulose membrane) with pores. The principle behind dialysis is usually diffusion, which is explained further. This membrane contains micro pores through which smaller molecules and ions (present along with the protein sample) move out (from region of higher concentration) and emerge in the dialysate outside the bag (to their region of lower concentration), across a concentration gradient, till equilibrium is achieved. To balance this, water/buffer molecules traverse into the dialysis bag, across a concentration gradient. However, the protein molecules which have sizes that are significantly greater than the membrane pore diameter, therefore cannot traverse out of the dialysis tubing and remain retained inside the dialysis bag. Figure 4 shows a typical set up for protein dialysis. Figure 4. Dialysis of proteins 8

9 As shown in Figure 4, the protein/enzyme solution is placed in a dialysis bag and immersed in a large volume of buffer that is stirred and maintained at about 4 C. During dialysis, the salt molecules will tend to diffuse out of the dialysis bag. If the buffer is changed several times, more or less all the salt will be removed from the protein solution. It is important to note that dialysis will result in an increase the volume of the enzyme solution, because water molecules from the buffer enter into the bag. It is therefore necessary to leave some space/gap at the top of the membrane tube as shown in Figure 3, to prevent it from bursting. 4. Summary In this lecture we learnt about: Protein purification is a multistep procedure. Fractionation helps to separate proteins into different fractions based on several properties such as size, charge etc. Early fractionation steps utilize the difference in protein solubility. Precipitation of proteins from a solution using high osmotic strength (i.e. salt solutions) is a common primary purification step. Ammonium sulphate is commonly used salt. The effectiveness of the different ions towards protein precipitation is given by Hofmeister series. Removal of salt used to precipitate the protein is first removed (commonly through dialysis) before subjecting it to subsequent purification steps. 9