Abstract There are many methods employed to precipitate proteins out of solution. In this experiment we manipulated many physical and chemical variables in order to achieve purification of a protein via precipitation. In the first part of the experiment we purified the protein casein by modifying it’s pH. In the second part of the experiment we manipulated the ionic strength of albumin in egg whites, in a process called salting out. By manipulating these chemical properties we were able to precipitate casein from skim milk and albumin from egg whites. The final product was weighed, and recorded.
Final mass of albumin per egg was 0. 142 grams. The final mass of casein from skim milk was 5. 522 grams. These methods were both successful at separating the protein out of the solution, but the technique that produced the highest percent yield was acidification and modification of a dielectric constant with ethanol. Intro Proteins can be precipitated out of solution. By modifying the physical and chemical properties, it is possible to selectively precipitate proteins. For example, proteins precipitate from solution when the pH of the solution equals the pI.
So by adding acid to reduce the pH to the value to the pI, we can achieve precipitation of specific proteins, depending on their individual pI’s. Another method for precipitating proteins is the salting-in and salting-out method. This process achieves protein separation by using the ionic strength and solubility differences associated with adding salt to the solution. The salt that is most commonly employed in this method is ammonium sulfate. A third variable that can be changed in order to precipitate a protein is heat. Heat application interferes with the noncovalent chemical bonds that keep protein in a folded conformation.
The fourth property that is manipulated for protein precipitation is the dielectric constant, which is a variable associated with Coulomb’s Law. Certain chemical compounds, such as ethanol have a lower dielectric constant, so adding them to solution containing protein increases the force of attraction between protein molecules. See Equation #1. Due to this increased affinity between the 2 proteins, they become so close together, that they stick to one another, and eventually precipitate out. In both parts of the experiment a combination of these techniques were utilized in rder to isolate the casein and albumin proteins. Materials and Method -100mLs skim milk-2 egg whites -distilled water-6 mL 1. 0 M acetic acid -pH meter-cheesecloth -centrifuge-ammonium sulfate -4 mL 10% HCl-centrifuge -20 mL 95% ETOH-20 mL acetone -Buchner funnel/filter paper-Buchner funnel/filter paper For part 1 of the experiment, combine 100 mL skim milk and 300 mL distilled water. 3. 9 mLs of 10% HCl is slowly added over a 10 minute time period. The solution is constantly stirred for 30 minutes. The solution then gets centrifuged. After the centrifuge, the supernatant is decanted, and 20 mLs ethanol was added.
The solution is finally vacuum-filtered in a Buchner funnel, and collected, and weighed. For the second part of the experiment 2 egg whites were added to 5. 6 mLs acetic acid. The solution was then ran through a double-layered cheesecloth. Ammonium sulfate was added. (25mLs) The solution was stirred for 30 minutes. The supernatant was collected after the solution was centrifuged, and the precipitate was discarded. One more mL of ammonium sulfate was added as we stirred, until the solution remained turbid. The supernatant was decanted, and the solution was centrifuged again.
After the final centrifuging, 20 mLs of acetone was added, and the compound was vacuum- filtered, collected, and weighed. Results and Discussion- In the first part of the experiment we used acid to lower the pH of skim milk. When the pH=pI, protein will precipitate out. The pH of milk is around 6. 5. The casein pI is 4. 6. By adding acid to the milk until the pH is reduced from 6. 5 to 4. 6, we successfully forced the protein to separate out. (See Equation #2) If skim milk had another protein that had a pI of 3. 5, we would have to add more acid in order to get that secondary protein to precipitate out with the casein.
At the isoelectric point the protein is uncharged and displays minimal water solubility. At low pH the protein carries a positive charge, at high pH the protein carries a negative charge. We would need the pH of the solution to be 3. 5 if we wanted to precipitate a protein out of it with as isoelectric point of 3. 5. This is a nice feature of proteins, because it allows us to selectively precipitate the proteins, when several are present. The final mass of casein recovered was 5. 522 grams. The percent yield was calculated to be 69. 025%. See Calculation #1) This is a reasonable percent yield of recovered casein from skim milk. Milk actually contains 3 main types of protein, casein, lactalbumins, and lactoglobulins. It is possible that the other 2 proteins did not precipitate out, and that’s why only approximately 70% was recovered. Ethanol and acetone are often used to precipitate protein. These solutions have lower dielectric constants. This method takes advantage of the forces of attraction between proteins. This force, associated with Coulomb’s Law, can be related to the charges of 2 separate proteins, the distance between them, and the dielectric constant. Equation #1) By reducing the dielectric constant, you increase the force of attraction between the two proteins. When the proteins are so attracted to one another, that they stick together, they will precipitate out of solution. The final mass of albumin per egg that we recovered was 0. 142 grams. The percent yield was calculated to be 2. 3%. (See Calculation #2) This is a very small percent yield. The low value may be attributed to the fact that we only used egg whites, and not the yolks. The majority of the albumin could be concentrated in the yolk. Additionally, the precipitation method may be less efficient than others.
Conclusion There are a variety of techniques used to precipitate protein out of solution. Selective protein precipitation is possible due to the proteins individual chemical and physical properties. Adjusting pH to equal the isoelectric point, is one method of selective protein precipitation. In the first part of this experiment, the pH was lowered with acid, and the dielectric constant was manipulated by adding ethanol. For the second part of the experiment, the pH was also lowered to the value of the isoelectric point, but the secondary technique employed was “salting out” the protein with ammonium sulfate.
Based solely on percent yield calculations, the variable modification that yielded the most protein was the acidification and dielectric constant. This may be partially due to the design of the experiment, however, because we did not include the protein found in the egg yolks, in that calculation. Additionally, the percent yield associated with the precipitation of casein from skim milk, may have been low because the total protein also included lactalbumins and lactoglobulins, which may not have precipitated out with the casein.