EXPERIMENT NO. 6C

ENZYME PURIFICATION

BY ISOELECTRIC 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 changing the pH of the solution.

Introduction

The solubility of protein depends on, among other things, the pH of the solution. Similar to the amino acids that comprise protein, protein itself can be either positively or negatively charged overall due to the terminal amine -NH2 and carboxyl (-COOH) groups and the groups on the side chain. It is positively charged at low pH and negatively charged at high pH. The intermediate pH at which a protein molecule has a net charge of zero is called the isoelectric point of that protein. In general, the net charge on the protein, either positive or negative, can interact with water molecules, meaning that it is more likely for a protein molecule to dissociate itself from other protein molecules, thus, more soluble. As a result, protein is the least soluble when the pH of the solution is at its isoelectric point.

When microorganisms grow in milk, they often produce acids and lowers the pH of the milk. The phenomenon of precipitation or coagulation of milk protein (casein) at low pH as milk becomes spoiled is one of the common examples of protein isolation due to changes in the pH.

List of Reagents and Instruments

A. Equipment

B. Reagents

Procedures

  1. Precipitation of Protein in Acidified Solution:
    • Add 5.0 g of casein to 200 ml of 1N NaOH solution.
    • Pipet 4 ml of the protein solution into a test tube.
    • While stirring, add the acid solution drop-wise to the alkaline protein solution from a graduated pipet or a buret until precipitates start to form. Stir thoroughly to avoid the localization of low pH spots in the solution. Note the volume of the acid solution added at the incipient of precipitation. Since precipitation is not an instantaneous process, let the test tube stand undisturbed for 30 minutes.
    • Repeat the same process for a series of test tubes, each containing 4 ml of the alkaline protein solution. To each test tube, add slightly less acid solution than the previous one so that a series of pH values can be established. Let each test tube stand for 30 minutes. Measure the pH of each solution and note the pH region around which the amount of precipitate is the maximum.
  2. Try out other proteins or enzymes available in the laboratory.
  3. Isolation of Protein Components from a Mixture:
    • If the student conclude that the individual protein component can be fractionally isolated, repeat the same procedures as in ammonium sulfate precipitation.

Questions

  1. Report the amount of precipitate as a function of the pH for each of the proteins tried.
  2. Justify why pH fractionation can/cannot be used to isolate different proteins.
  3. Comment on ways to improve the experiment.

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Enzyme Purification By Isoelectric 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