Enzymology BCH3108 Amm Sulphate PDF

3/7/2024 SALT AND SOLVENT FRACTIONATION OF ENZYMES...

3/7/2024 SALT AND SOLVENT FRACTIONATION OF ENZYMES  Salt fractionation by precipitation of enzyme is a common method to ENZYMOLOGY BCH3108 – Amm Sulphate purify enzyme. It is usually employed early. All rights reserved- Prof Yunus Prof. Dr. Mohd Yunus Abd. Shukor Dept. of Biochemistry Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia 43400 UPM Serdang Selangor, Malaysia. 1 1 2 CONCEPT  Salt such as ammonium sulphate when added to a solution containing proteins (at least 1 mg/ml concentration) will disrupt the water cage surrounding the protein and causes reversible denaturation by ‘opening up’ the hydrophobic portion of proteins to aqueous environment.  Since hydrophobic proteins tend to aggregate together to ‘run away’ from water they will coalesce and aggregate and the results is precipitation of proteins. 2 3 3 4  3/7/2024 mechanism Mechanism  According to scopes,  Certain proteins will be disrupted at low concentration ‘saturation’ of salt. Other  The mechanism of salting out as a precipitation method is generally through its proteins get disrupted at higher salt saturation. ability to affect the electrostatic and nonpolar properties of proteins in a reversible  Using this advantage, protein can be segregated into several portions using different manner. At concentrations of above 0.2 M, salts not only neutralize the electrostatic saturations or ‘cuts’ of ammonium sulphate. forces on the protein surface but also affect the tertiary and quaternary structure of  Amongst the many candidates for the salting out method, ammonium sulphate is proteins, exposing the hydrophobic interior to the environment and making proteins the most often used salt in the “salting out” fractionation method (Scopes, 1988). less soluble (Scopes, 1988).  Separating proteins from a crude extract by "salting out" using ammonium sulphate  The hydrophobic groups on the proteins get exposed to the atmosphere, attract is a convenient purification step due to the many advantages such as other protein hydrophobic groups and get aggregated. Protein precipitated will be large enough to be visible 4 5 5 6 Mechanism Why ammonium sulphate  Salting In and Salting Out.  at its saturated point (760 g/L), most proteins are precipitated and thus most During the salting in process, salt molecules increase the proteins can be fractionated, solubility of proteins by reducing  ammonium sulphate suspension can act as bacteriostatic agent, the electrostatic interactions  it is able to accommodate large amount of sample and between protein molecules.  most enzyme preparations in saturated ammonium sulphate suspensions could be  As the salt concentration is increased, protein-protein stored at long period of time at 4oC. interactions become more  When dissolved, minimal heat is produced compared to other salts energetically favorable than  If this method is to be followed by another technique such as ion exchange protein-solvent interactions and chromatography, or you need to determine protein and enzyme assay, then the salt the proteins precipitate from needs to be removed preferably by dialysis. solution. 5 6 7 8  3/7/2024 Amounts of solid ammonium sulfate required to change the % saturation of 100 mL of solution Ho to use table  The table below is used to determine how much ammonium sulfate (in grams) must  If the initial concentration is 0% saturation (crude), so in the first column I find the be added to100 mL of solution to change from any particular % saturation to any box called "0 %." The desired final concentration is 20% saturation, so in the top other % saturation. row I find the box called “20 %."  To use the table, start by finding the entry in the first column that gives the initial %  The value at the intersection of this row and column is “13.4" so I would add 13.4 g saturation of your solution. (For example, this would be the first entry, 0%, before to 100 mL of sample. Since I am using only 25 mL, I would add only a quarter as any ammonium sulfate has been added.) much, or 3.35 g.  Then find the entry in the top row that shows the desired final % saturation of  If the initial concentration is 40% saturation, so in the first column I find the box ammonium sulfate for the solution. called "40 %." The desired final concentration is 80% saturation, so in the top row I  The value at the intersection of that row and column is the amount of solid find the box called "80 %." ammonium sulfate that must be added to 100 mL of solution to change from the  The value at the intersection of this row and column is "29.6" so I would add 29.6 g initial to final % saturation. to 100 mL. Since I am using only 25 mL, I would add only a quarter as much, or 7.4 g. 7 8 9 10 Init % 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Dry practical 0 10.6 13.4 16.4 19.4 22.6 25.8 29.1 32.6 36.1 39.8 43.6 47.6 51.6 55.9 60.3 65.0 69.7 5 7.9 10.8 13.7 16.6 19.7 22.9 26.2 29.6 33.1 36.8 40.5 44.4 48.4 52.6 57.0 61.5 66.2 10 5.3 8.1 10.9 13.9 16.9 20.0 23.3 26.6 30.1 33.7 37.4 41.2 45.2 49.3 53.6 58.1 62.7 15 2.6 5.4 8.2 11.2 14.1 17.2 20.4 23.7 27.1 30.6 34.3 38.1 42.0 46.0 50.3 54.7 59.2 1. Measure the volume of the protein solution, pour it into a chilled beaker with a 20 0 2.7 5.5 8.3 11.3 14.3 17.5 20.7 24.1 27.6 31.2 34.9 38.7 42.7 46.9 51.2 55.7 magnet bar and place it in an ice bath or at 4°C on a slow stirrer. 25 0 2.7 5.6 8.4 11.5 14.6 17.9 21.1 24.5 28.0 31.7 35.5 39.5 43.6 47.8 52.2 30 0 2.8 5.6 8.6 11.7 14.8 18.1 21.4 24.9 28.5 32.3 36.2 40.2 44.5 48.8 2. Use the solid form of ammonium sulphate that has been grounded, calculate the 35 0 2.9 5.7 8.7 11.8 15.1 18.4 21.8 25.8 29.6 32.9 36.9 41.0 45.3 required amount of ammonium sulphate by referring to the table number. The 40 45 0 2.9 0 5.8 3.0 8.9 5.9 12.0 9.0 15.3 12.3 18.7 15.6 22.2 19.0 26.3 22.6 29.6 26.3 33.5 30.2 37.6 34.2 41.8 38.3 amount of ammonium sulphate powder added will be different according to the 50 0 3.0 6.0 9.2 12.5 15.9 19.4 23.5 26.8 30.8 34.8 volume of your sample. 55 0 3.1 6.1 9.3 12.7 16.1 20.1 23.5 27.3 31.2 3. Add the chilled ammonium sulphate powder slowly and gently into your chilled 60 0 3.1 6.2 9.5 12.9 16.8 20.1 23.9 27.9 protein solution in small portions but steadily with thorough mixing. 65 0 3.2 6.3 9.7 13.2 16.8 20.5 24.4 70 0 3.2 6.5 9.9 13.4 17.1 20.9 4. Stir slowly for 15-20 min to fully equilibrate at 4°C. Do not allow clumps to form. 75 0 3.3 6.6 10.1 13.7 17.4 Allow the salt to dissolve before adding the next portion. As the reaction produces 80 0 3.4 6.7 10.3 13.9 heat, the ice jacket is important. If the reaction can be carried out in a cold room- 85 0 3.4 6.8 10.5 90 0 3.4 7.0 the better. 10 95 0 3.5 100 0 11 12  3/7/2024 Dry practical 5. Recover precipitate by centrifuging the solution at 15,000 ×g for 10 min at 4°C to pellet out protein. Solutions highly saturated in ammonium sulphate are quite dense, and it can be difficult to pellet the precipitate. 6. Collect the precipitate by carefully transferring out as much supernatant as possible. 7. Add more saturated ammonium sulphate or solid ammonium sulphate to the supernatant to make next concentration, repeat stirring and centrifugation. 8. Dissolve pellets in 0.1 M Phosphate pH 7.5 to determine enzyme activity and protein concentration using enzyme assay and Bradford assay, respectively. 12 13 14 Dry practical-dialysis Dry practical-dialysis 9. Once you have determined that you have enzyme activity in your protein pellet, proceed with the next step in enzyme purification. Depending on what is your next step, it is necessary to remove the ammonium sulphate from your protein solution. The simplest way to do this is through dialysis. 10. the protein solution is placed in a bag of the selectively permeable membrane (e.g. cellophane). 11. Immersed the sample solution (e.g. 5-10 mL) from the 40 to 60% ammonium sulphate fraction suspension in a large volume (1 L) of 50 mM Tris-Cl pH 7.0 (Buffer A) that is stirred and maintained at about 4°C for 1 hour. 12. Notes that the membrane has pores that will permit small molecules such as ammonium and sulphate ions to cross, and hence equilibrate in the larger volume Ammonium sulphate fraction is usually a bit cloudy at the start of the dialysis. For a 100 mL volume dialyze of buffer outside, while not permitting large protein molecules to cross. in 1 L of buffer, equilibration is complete after about 2 hours. Change the buffer, and this should quicken 13 dialysis. 14 15 16  3/7/2024 The balance sheet for the ammonium sulphate fractionation Specific NH4SO4 Activity Fold Fraction Total Protein (Unit/mg Total Activity Yield % Purification Crude (mg) protein) (Units) 5,000 6 30,000 100.00 1.00 Crude 600 4 2,400 8.00 0.67 0-20 950 4 3,800 12.67 0.67 20-30 560 10 5,600 18.67 1.67 30-40 750 39 29,250 97.50 6.50 40-50 350 10 3,500 11.67 1.67 50-60 213 7 1,491 4.97 1.17 60-70 70-100 112 0 0 0 0 Protein and enzyme activity for each ammonium sulphate fractions. 15 16 17 18 Notes Note  The 40-50% fraction gave the highest enzyme yield as well as fold purification. This  The cumulative yield of enzyme percentage from the ammonium sulphate fraction should be used in subsequent fractionation fractionation exceeds the yield in crude. 153.48% (not possible numerically!!)  Total activity is calculated as a specific activity x total protein.  The probable reason is that an enzyme inhibitor has been removed during  The crude enzyme preparation is assigned as 100% yield or starting amount of fractionation from the crude, increasing the activity in the new fraction. enzyme. This value in percentage is calculated as (Total activity in X fraction/Total  Further detail do read activity in crude) x 100%.  Yield is expected to be lower as more and more fractionation methods are https://upmpress.com.my/product/basic-enyzme-purification- employed to get pure enzyme. with-work-examples/  Fold purification denotes how pure is the enzyme preparation compared to crude. Calculated as the Specific activity of Fraction X/specific activity in crude. An increase in fold purification suggest that many impurities have been removed but is not important when the objective is to get a pure enzyme. 17 18 19 20