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Exercise 4.1
Isolation of Proteins
Groupmates:
Desiree Joy Cerico
Ma. Kriselle Ornales
Mary Ranzelle Pasang
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.
Observations
Yellowish viscous liquid
Formation of white clumps
White viscous clumps
Yellowish opaque liquid
Formation of bubbles, cloudy yellow
liquid
White bubbles with white precipitate
White cloudy liquid
White, milky liquid with bubbles
Yellowish liquid
White bubbles with white precipitate
White, milky liquid
White precipitate
words, the higher the ammonium sulfate concentration (salt), the least soluble the
proteins become in the solution, causing them to precipitate out.
pH
2.7
4.7
6.7
Observations
Clear solution
Cloudy
Clear solution
volume of egg
(weight of dri ed precipitate)
3.397 g
40 mL
References:
Bettelheim, F. (2007). Introduction to General, Organic and Biochemistry.
Brooks/Cole by Thomson Learning.
Brown, C. (2005). Introduction to Biochemistry. McGraw-Hill International.
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