Post-laboratory Report on Exercise 4.1
Isolation of Proteins
Vikki Anne R. Cedo CHEM 160.1 - 3L 2nd Semester 2014-2015
Groupmates:
Desiree Joy Cerico Ma. Kriselle Ornales Mary Ranzelle Pasang
Ms. Korina Vida G. Sinad Laboratory Instructor
Exercise 4.1 Isolation of Proteins
Proteins are by far the most important of all biological compounds. Many types of this biological compound exist, and they perform a variety of functions including structure, transport/movement, hormones, protection, storage and regulation. This diversity of functions make them the most studied biological compound. Many isolation and purification techniques were successfully done by scientists as well as techniques for determining the quality and quantity of the isolate.
Making use of the knowledge of solubility and isoelectric point of proteins, protein precipitation method was established. Proteins are isolated from a mixture of soluble substances. Knowing the solubility of your protein of interest, you can now easily precipitate out your protein from the solution by designing a precipitation technique (choosing what reagent you'll add to the solution). Salting out process and isoelectric precipitation are the two most commonly used techniques.
Salting out is a technique wherein salts such as ammonium sulfate or sodium sulfate are used to precipitate out proteins by adding them into the solution. Ammonium sulfate is the one which is widely used for protein precipitation by salting out because it is highly soluble, available in the highest purity level and does not change the pH to extremes and it does not denature proteins; it also protects solution from bacterial growth. During ammonium sulfate precipitation, the salt has to be added in small amounts with constant stirring so as to avoid accumulating high concentration of salts. When large amount of salt is added to an aqueous solution of proteins, the salt will require more amount of water for its dissolution. This will lead to competition for water molecules then protein hence addition of salts take up water molecule from proteins. The ionic interactions between water molecules and protein are reduced and as a result hydrophobic interactions dominate. The hydrophobic amino acid patches present in all the proteins attract each other and form aggregates. These aggregates are nothing but the proteins in the form of precipitates.
Isoelectric precipitation is another protein isolation technique. This technique makes use of the knowledge of the pH of the solution and the IpH or isoelectric point of the protein you desire to isolate. Changes in pH of the solution change the ionization state of the proteins' functional groups and its net charge. When the pH of the solution is equal to the isoelectric point of the protein, it is the time when the protein becomes least soluble and therefore precipitates out of the solution. The solubility of the protein increases if the pH of the solution is somewhere to the right or to the left (higher or lower) of the isoelectric point.
Protein solubility depends on numerous factors. It is observed that at low concentration of the salt, the solubility of the protein increases by a small percentage. This phenomenon is called salting in. At high concentrations of the salt, the solubility of the proteins drop dramatically and this is called salting out because the proteins precipitate out of the solution. In salt precipitation, the anions appear to be more important. pH, temperature and protein concentration affect ammonium sulfate precipitation of proteins to large extent. Higher ammonium sulfate is required for precipitating highly soluble proteins.
Ammonium sulfate can be used for precipitation of total proteins at ~90% saturation or for differential precipitation level of proteins using different saturation of salts. Up to 20 % saturation, ammonium sulfate precipitate particulate materials, and preaggregated and very high molecular weight proteins and at 90 % saturation it precipitates almost all proteins.
Table 4.1.1. Preparation of crude egg albumin by ammonium sulfate precipitation.
Steps Observations
Filtered undiluted egg white Yellowish viscous liquid Addition of 1 M acetic acid Formation of white clumps Removal of precipitate| Precipitate: White viscous clumps | Filtrate: Yellowish opaque liquid After addition of 7.26 g ammonium
sulfate powder
Formation of bubbles, cloudy yellow liquid
Filtration | Residue: White bubbles with white precipitate | Filtrate: White cloudy liquid
After addition of 3.9 g ammonium sulfate powder
White, milky liquid with bubbles After 30 minutes of standing Yellowish liquid
Filtration | Residue White bubbles with white precipitate | Filtrate White, milky liquid
After air drying White precipitate
Mass of filter paper: 1.707 g
Mass of filter paper + crude egg albumin: 3.397 g Mass of crude egg albumin: 1.69 g
Increasing the concentration of ammonium sulfate in the experiment to obtain or isolate proteins is essential because the higher the concentration of the salt, the more proteins that it will be able to precipitate out of the solution. In other
words, the higher the ammonium sulfate concentration (salt), the least soluble the proteins become in the solution, causing them to precipitate out.
Table 4.1.2. Isoelectric precipitation of casein.
Test tube no. pH Observations
1 2.7 Clear solution
2 4.7 Cloudy
3 6.7 Clear solution
Calculation of yield (60% ammonium sulfate precipitate):
=
volume of egg
(
weight of dri ed precipitate)
¿
¿
=3.397 g
40 mL
= 0.084925 x 100 = 8.4925 or 8.50%In the isoelectric precipitation of casein, only test tube number 2 was observed to have a cloudy mixture and the other two test tubes contain clear solutions. Test tube number 2 contains acetate buffer and has a pH of 4.7, casein, distilled water and 1M NaOH and 1M CH3COOH, while test tube number 1 contains
glycine-HCl buffer casein, distilled water, 1M NaOH and 1M CH3COOH with a pH of
2.7and test tube 3 contains phosphate buffer, casein, distilled water, 1M NaOH and 1M CH3COOH with a pH of 6.7. After adding the solution of casein, distilled water,
1M NaOH and 1M CH3COOH to the buffers, only test tube number 2 was observed to
exhibit cloudiness. It can be concluded that the IpH of casein is 4.7 since it where the mixture got cloudy. At pH 2.7 and 4.7 (test tubes 1 and 3), it can be concluded that casein is soluble because pH 2.7 is lower than its isoelectric point and 6.7 is higher than the isoelectric point. The theoretical IpH of casein is 4.6. It is the pH at which the protein is least soluble. Test tube no. 2. exhibited a cloudy mixture so it can be concluded from the result of the experiment that casein's IpH is at 4.7. Casein precipitated out of the solution because its solubility is decreased at that pH, so they aggregated together and precipitated out.
References:
Bettelheim, F. (2007). Introduction to General, Organic and Biochemistry. Brooks/Cole by Thomson Learning.
Education Portal - Life Science. (n.d.). Retrieved March 1, 2015, from E-learn Biotechnology: http://www.elearnbiotechnology.com/practical_project.php? id=9&type=practical&chapter=9
Ophardt, C. (2003). Virtual Chembook. Retrieved February 21, 2015, from Elmhurst College: http://elmhurst.edu/~chm/vchembook/568denaturation.html
Wang, N. S. (n.d.). Chemical & Biomolecular Engineering Official Site. Retrieved March 2, 2015, from Maryland University Department of Chemical & Biomolecular Engineering: http://www.eng.umd.edu/~nsw/ench485/lab6c.htm
