Experiment 5 Isoelectric pH of Casein

EXPERIMENT 5
ISOLATION OF CASEIN
FROM MILK USING
ISOELECTRIC (pH)
PRECIPITATION METHOD

Structure

5.1 Introduction
5.4 Procedure
Expected Learning Outcomes
5.5 Results
5.2 Principle
5.6 Precautions
5.3 Materials Required

5.1 INTRODUCTION

In the previous experiments you have performed the estimation of protein

concentration by using different methods. In this experiment you will perform
isolation of milk protein i.e., casein. Electrostatic charge is one of the
important factors that contribute to protein solubility. The solubility of proteins
in aqueous buffers depends on the distribution of hydrophilic and hydrophobic
amino acid residues on the protein surface. Proteins that have high
hydrophobic amino acid content on the surface have low solubility in an
aqueous solvent. Charged and polar surface residues interact with ionic
groups in the solvent and increase their solubility. The net charge of a protein
molecule is the arithmetic average of all charges. At a certain solvent pH, the
protein net charge will be zero this is called the isoelectric point.

At a solution pH that is above the pI of the surface of protein is predominantly

negatively charged and therefore like charged molecules will exhibit repulsive
forces. On the other hand, surface of the protein is predominantly positively
charged at a solution pH that is below the pI, and repulsion between proteins
occurs. Therefore, protein will be soluble at this pH. However, at the pI the
negative and positive charges are eliminated, repulsive electrostatic forces are
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BBCCL-106 Proteins

reduced and the dispersive forces will cause aggregation and precipitation. It
is important to note that, pI of most proteins ranges between the pH 4 to 6.

You might be surprised to know that, the solubility of casein is not affected by
heat because it does not contain disulphide bonds and lack the tertiary
structure. However, the solubility of casein depends greatly on the pH of the
medium. At isoelectric point (pH 4.8) of casein, the solubility of protein is
minimum, but increases with increasing acidity or alkalinity.

You are advised to watch the video available at the following YouTube link
before going to start the experiment
https://www.youtube.com/watch?v=HN4jD2MCKfg.

Table 5.1: Isoelectric points of few important proteins

Isoelectric point Protein

(pH)

7.0 Myoglobin

6.8 Haemoglobin

4.8 Gelatin

4.9 BSA

4.8 Casein

4.6 Egg albumin

1.0 Pepsin

Expected Learning Outcomes

 describe the role of pH in precipitating casein;

 explain the importance of pH and pI on protein solubility; and
 demonstrate the use of centrifuge and pH meter.

5.2 PRINCIPLE
The isoelectric point of a protein is the pH where the net charge on the protein
is zero. Proteins tend to aggregate and precipitate at their respective pI values
due to lack of electrostatic repulsion that keep them apart. Proteins have
different pI values owing to their amino acid composition (i.e., relative numbers
of anionic and cationic groups), and thus they can be separated by adjusting
the pH of a solution. When the pH is adjusted to the pI of a particular protein, it
precipitates leaving the other proteins in solution. Maximum precipitation can
be obtained at the isoelectric point by addition of some reagents such as,
ethanol which dehydrates the molecule and allow neutralization of charge.

The pH of any solution can be calculated from following Handerson-

26 Hasselbalch equation. (Refer unit-2 of BBCCT-101 for more details).
Experiment 5 Isoelectric pH of Casein

pH = pKa + log {(casein acetate sodium) ÷ (acetic acid)}

5.3 MATERIALS REQUIRED

Glassware and Equipment’s: Test Tubes, Test Tube Stand, Pipettes,
Spatula, Whatman number 1 filter paper, Glass funnel, Petri dish, Watch
glass, Filtration Unit, Weighing Balance, Centrifuge and Ph Meter.

Chemicals and Reagents: 0.5 N Hydrochloric acid, Ethanol, diethyl ether

Sample: Cow or Buffalo milk

5.4 PROCEDURE
Perform the following steps as instructed:
i. Skimming of milk: Take 100 ml of milk and centrifuge at 4000 rpm for
about 25 minutes at room temperature. Later remove the upper fat or
cream layer using a clean spatula.
ii. Transfer the skimmed milk into a 500 ml glass beaker followed by
equal volumes of distilled water and mix well. Take a note of the pH.
iii. Add 0.5 N HCl drop by drop using burette with constant stirring until the
pH reaches to 4.8. At this pH (Isoelectric point) casein starts
precipitating.
iv. Allow the mixture for 30 minutes at room temperature. This completes
the precipitation.

v. Later, decant the supernatant (upper clear liquid) and filter the
suspension (precipitate) carefully using Whatman No. 1 filter paper
(fixed on glass funnel) under mild suction pressure using a pump.

vi. The precipitate is washed with washed 2-3 times with distilled water.
Followed by two washes each with 100 ml diethyl ether and ethanol.

vii. Shake the test tubes and wait for 10 minutes.

viii. Observe and write down the results use “+” and “-” to express the
quantity of precipitation.
ix. Transfer and spread the pellet uniformly on the dry watch glass or petri
dish and allow it to dry at room temperature for overnight.
x. Weigh the amount of casein, calculate and record percent the yield.

5.5 RESULTS
The amount casein obtained from 100 ml of milk is________gm

Discussion:

The following are the hydrophilic amino acids: Arginine, Asparagine,

Aspartate, Glutamine, Glutamate, Histidine, Lysine, Serine and Threonine)
and hydrophobic amino acids: Valine, Tyrosine, Tryptophan, Proline,
Phenylalanine, Methionine, Leucine, Isoleucine, Cysteine and Alanine
respectively.
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BBCCL-106 Proteins

5.6 PRECAUTIONS
1. Use cold diethyl ether and keep the bottle away from flame.
2. Calibrate the pH meter with standard buffer before you start with the
experiment. Make sure that the pH meter is working properly.
3. Wash the pH electrode thoroughly with distilled water and wipe the
electrode smoothly. Care should be taken to ensure that lint free tissue
paper is used to wipe the pH electrode.
4. Do not immerse the electrode into the solution deeper than 20 mm.
5. There will be fluctuations in pH reading when the electrode is dipped
into the solution, so make sure to wait long enough for the reading to
get stabilize.

SAQ

1. Define isoelectric point.

2. What is zwitter ion?
3. Explain the role of isoelectric point on protein precipitation.

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