salting in, salting out, and dialysis of proteins

KSU - College of Science - Department of Biochemistry BCH 233, Zaenab Alzahrani, 2nd semester, 2009-2010

Page 1 of 5

Physical Biochemistry Lab

Experiment no. 5:

Salting in, salting out, and dialysis of proteins

Objective:

To learn the techniques of isolation of proteins on basis of their solubility in water.

Introduction and principle:

Proteins are the most abundant biological macromolecules occurring in all cells and all parts

of cells. Our understanding of protein structure and function has been derived from the study

of many individual proteins. To study a protein in detail, the researcher must be able to

separate it from other proteins and must have the techniques to determine its properties. The

necessary method comes from protein chemistry, a discipline as old as biochemistry itself and

one that retains a central position in biochemical research. A pure preparation is essential

before a protein's properties and activities can be determined. Given that cells contain

thousands of different kinds of proteins, how can one protein be purified? Methods for

separating proteins take advantage of properties that vary from one protein to the next. The

source of a protein is generally tissues or microbial cells. The first step in any protein

purification procedure is to break open these cells, releasing their proteins into a solution

called a crude extract. Once the extract is ready, various methods are available for purifying

one or more of the protein it contains. Commonly, the extract is subjected to treatments that

separate the proteins into different fractions based on a property such as size or charge, a

process refered to as fractionation. Early fractionation steps in purification utilize differences

in protein solubility.

Because a protein contains multiple charged groups, its solubility depends on the

concentrations of dissolved salts, the polarity of the solvent, the ph, and the temperature.

Some or all of these variables can be manipulated to selectively precipitate certain proteins

while others remain soluble. The solubility of a protein at low ion concentrations increases as

salt is added, a phenomenon called "salting in". The additional ions shield the protein's

multiple ionic charges, thereby weakening the attractive forces between individual protein

molecules (such forces can lead to aggregation and precipitation). However, as more salt is

added, particularly with sulfate salts, the solubility of protein again decreases. This "salting

out" effect is primarily a result of the competition between the added salt ions and the other

dissolved solutes (protein molecules) for molecules of solvent (water). At very high salt

concentrations, so many of the added ions are solvated that there is significantly less bulk

solvent available to dissolve other substances, including proteins.


Page 2 of 5

Since proteins precipitate at different salt concentrations, salting out is the basis of one of

most commonly used protein purification procedures. Adjusting the salt concentration in a

solution containing a mixture of proteins to just below the precipitation point of the protein to

be purified eliminates many unwanted proteins from the solution. Then, after removing the

precipitated proteins by filtration or centrifugation, the salt concentration of the remaining

solution is increased to precipitate the desired protein. The precipitation of desired protein is

then dissolved in water to make a solution of this protein. This procedure results in a

significant purification and concentration of large quantities of protein. Ammonium sulfate,

(NH4)2SO4 , is the most commonly used salt for salting out proteins because its large solubility

in water, its relative freedom from temperature effects, and it has no harmful effects on most

of the proteins. The most effective pH region for salting out of the desired protein is at its

isoelectric point because the protein is least soluble when its net charge is zero.

Effect of salt concentration on protein solubility


Page 3 of 5

A solution containing the protein of interest often must be further altered before subsequent

purification steps are possible. Dialysis is one of the common operations in biochemistry to

separate dissolved molecules by passing through a semi-permeable membrane according to

their molecular dimensions.

Semi-permeable membrane is containing pores of less than macromolecular dimensions.

These pores allow small molecules, such as those of solvents, salts, and small metabolites, to

diffuse across the membrane but block the passage of larger molecules. Cellophane

(cellulose acetate) is the most commonly used dialysis material although many other

substances such as nitrocellulose and collodion are similarity employed. So, dialysis is a

method in which an aqueous solution containing both macromolecules and very small

molecules which are placed in a dialysis bag which is in tern placed in a large container of a

given buffer or distilled water. Thus small solute molecules freely pass through the

membrane, and after several hours of stirring the equilibrium will reach (the concentration

inside and outside the bag are the same). Thus, at equilibrium the concentration of small

molecules outside and inside the bag is the same while the macromolecules remain inside the

bag. During dialysis the external fluid should be changed in order to reach the required

composition inside the dialysis bag. There are three factors that affecting the rate of dialysis:

the first is the concentration differences of that molecules between the internal and external

solution (which is the driving force for the movement of the molecules). The second is mixing

on both sides of dialysis membrane will increase the rate of movement prevent the small

particles on the side of low concentration. The third is dialyzable particles size versus pore

size of the membrane, substances that are very much smaller than the pore size will reach

equilibrium faster than substances that are only slightly smaller than the pores.

The main point to be noted is that there is a rapid initial drop in dialysis process followed by a

slow approach to equilibrium.

In this experiment myoglobin will isolated from skeletal muscle by salting out technique which

discards up to 75% of the crude proteins in the protein purification process.

The isoelectric point of the myoglobin is 7 and it is precipitate at salt (ammonium sulfate)

concentration of 55%. The ammonium sulfate used in salting out procedure will removed by

dialysis "desalting".


Page 4 of 5

Materials and apparatus:

1. Skeletal muscle

2. Distilled water

3. Ammonium hydroxide solution (2M)

4. Ammonium sulfate salt (solid)

5. Balance

6. Waring blender

7. Centrifuge tubes

8. Pasteur pipette

9. Centrifuge

10. pH meter

11. Measuring cylinder (10 ml)

12. Beaker (50 ml)

13. Dialysis bag

14. Dialysis tank

Method:

1) Cut skeletal muscle (100g) into small pieces and homogenize for 10 minutes in 100 ml

of distilled water at room temperature in a waring blender.

2) Divide the homogenate in 5 equal parts in centrifuge tube.

3) Allow the homogenate to stand for 20 min.

4) Centrifuge at 2000 rpm for 10 min. at 4C.

5) Discard the residue and adjust the pH of the supernatant to 7 by the addition of

(NH4)2SO4 (2M).

6) Measure the volume of the sample (supernatant).


Page 5 of 5

7) Calculate the required of ammonium sulfate salt needed to saturate the solution 35%

using ammonium sulfate nomogram.

8) Add the required salt to the solution slowly with small quantities and mix well

continuously after each addition.

9) After the addition is completed and the salt is completely dissolved, centrifuge at 3500

rpm for 10 min.

10) Discard the pellet and measure the volume of the supernatant.

11) Calculate the required salt in gram needed to saturate the solution 55% using

ammonium sulfate nomogram.

12) Add the required salt to the solution slowly with small quantities and mix well

continuously after each addition.

13) Centrifuge for 10 min. at 3500 rpm.

14) Discard the supernatant and dissolve the pellet in 10 ml dis. water and place it in

dialysis bag overnight.

Questions:

1) What do you understand by the terms:

a. Salting in

b. Salting out

c. Dialysis

2) Why ammonium sulfate is commonly used for "salting out" process?

* Figure 5-5 obtained from Fundamentals of Biochemistry life at the molecular level by Voet & Voet

* Figure 5-14 obtained from Biochemistry by Voet & Voet

salting in, salting out, and dialysis of proteins

Documents