EXPERIMENT NO. 6B
BY ACETONE PRECIPITATION
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
Table of Contents
To recover proteins/enzymes from a solution by adding acetone.
The solubility of protein depends on, among other things, the
dielectric constant of the solution. In general, solvent
molecules with large dielectric constants, e.g. water and
dimethylsulphoxide, can stabilize the interaction between
themselves and protein molecules and favor the dissolution of
protein. On the other hand, organic solvents with small
dielectric constants, e.g. acetone and methanol, discourage the
dispersion of protein molecules in the media. Thus, the
solubility of proteins can be lowered and precipitation can be
induced by lowering the effective dielectric constant of the
media. This is commonly achieved by adding a water-soluble
solvent such as acetone to an aqueous solution of protein.
Acetone had the advantage that it is relatively inexpensive and
is available in in a pure form with few contaminants that may
inhibit or poison the enzyme. It is also frequently used in
List of Reagents and Instruments
- Test tubes
- Graduated cylinder
- Filtration devices
- Protein solution, 5.0 g/l (albumin, gelatine, casein)
- Enzymes solution, 10 g/l (alpha-amylase, protease)
- Precipitation of Protein from Individual Protein Solution:
- Pipet 4 ml of the protein or enzyme solution into a test tube.
- While stirring, add acetone drop-wise to the protein solution
from a graduated pipet or a buret until precipitates start to
form. Vigorous stirring and slow acetone addition rate will
avoid the localized high concentration of acetone.
- Effect of Temperature on Enzyme Isolation:
- Perform Step for enzyme solutions 1 at 0ºC and compare the
- Isolation of Protein Components from a Mixture:
- Repeat the same procedures as in ammonium sulfate precipitation.
Most of the enzyme/protein separation and purification work is
conducted at low temperatures, and acetone precipitation is no
exception. In practice, it is routinely carried out at a
temperature below 0ºC.
- Calculate the volume fraction of acetone at the onset of
precipitation for each protein solution. Calculate the protein
yield for each case by following the same procedure as in
ammonium sulfate precipitation.
- How did the temperature affect the enzyme activities? How
would you extend this conclusion to the other proteins tried in
- Answer the same set of questions as in ammonium sulfate
- Comment on ways to improve the experiment.
Return to Prof. Nam Sun Wang's Home Page
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Enzyme Purification By Acetone Precipitation
Forward comments to:
- Nam Sun Wang
- Department of Chemical & Biomolecular Engineering
- University of Maryland
- College Park, MD 20742-2111
- 301-405-1910 (voice)
- 301-314-9126 (FAX)