EXPERIMENT NO. 6B

ENZYME PURIFICATION

BY ACETONE PRECIPITATION

Prepared by
Nam Sun Wang
Department of Chemical & Biomolecular Engineering
University of Maryland
College Park, MD 20742-2111
ENCH485

Table of Contents

Objectives

To recover proteins/enzymes from a solution by adding acetone.

Introduction

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 sterol extraction.

List of Reagents and Instruments

A. Equipment

B. Reagents

Procedures

  1. 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.
  2. Effect of Temperature on Enzyme Isolation:
    • Perform Step for enzyme solutions 1 at 0ºC and compare the enzymatic activities.
  3. Isolation of Protein Components from a Mixture:
    • Repeat the same procedures as in ammonium sulfate precipitation.

Discussions

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.

Questions

  1. 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.
  2. How did the temperature affect the enzyme activities? How would you extend this conclusion to the other proteins tried in this experiment?
  3. Answer the same set of questions as in ammonium sulfate precipitation.
  4. Comment on ways to improve the experiment.

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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)
e-mail: nsw@umd.edu