Enzyme Purification by Isoelectric Precipitation

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.

                                        The isoelectric point (pI) is the pH of a solution at which the net primary charge of a protein becomes zero. At a solution pH that is above the pI the surface of the protein is predominantly negatively charged and therefore like-charged molecules will exhibit repulsive forces. Likewise, at a solution pH that is below the pI, the surface of the protein is predominantly positively charged and repulsion between proteins occurs. However, at the pI the negative and positive charges cancel, repulsive electrostatic forces are reduced and the attraction forces predominate. The attraction forces will cause aggregation and precipitation. The pI of most proteins is in the pH range of 4-6. Mineral acids, such as hydrochloric and sulfuric acid are used as precipitants. The greatest disadvantage to isoelectric point precipitation is the irreversible denaturation caused by the mineral acids. For this reason isoelectric point precipitation is most often used to precipitate contaminant proteins, rather than the target protein. The precipitation of casein during cheesemaking, or during production of sodium caseinate, is an isoelectric precipitation.
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

• Test tubes
• Graduated cylinder
• Pipets
• Balance
• Centrifuge
• Filtration devices

B. Reagents

• Protein solution, 5.0 g/l (albumin, gelatine, casein)
• Enzymes solution, 10 g/l (alpha-amylase, protease)
• NaOH or KOH solution, 1N
• Acetic Acid solution, 0.1N

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

Sources:

http://terpconnect.umd.edu/~nsw/ench485/lab6c.htm
http://en.wikipedia.org/wiki/Protein_precipitation#Isoelectric_point_precipitation