Isolation and identification of casein
Adapted from Bettelheim
OBJECTIVE:
To isolate casein from milk and perform chemical tests to identify proteins.
BACKGROUND:
Casein is the most important protein in milk. It functions as a storage protein,
fulfilling nutritional requirements. Casein can be isolated from milk by acidification to
bring it to its isoelectric point. At the isoelectric point, the number of negative charges on
a protein equals the number of positive charges. Proteins are least soluble in water at their
isoelectric points because they tend to aggregate by electrostatic interactions. The
positive end of one protein molecule attracts the negative end of another protein
molecule, and the aggregates precipitate out of solution.
On the other hand, if a protein molecule has a net positive charge (at low pH or
acidic condition) or a net negative charge (at high pH or basic condition), its solubility in
water is increased.
+ −
H
+
NH 3CHRCOOH ←# # + NH 3CHRCOO− #OH#→ NH 2CHRCOO− + H 2O
#
more soluble least soluble more soluble
€ In the first part of this experiment, you will isolate casein from milk, which has a
pH of about 7. Casein will be separated as an insoluble precipitate by acidification of the
milk to its isoelectric point (pH = 4.6). The fat that precipitates along with casein can be
removed by dissolving it in alcohol. Alternatively, using skim milk leaves negligible
amounts of fat in the precipitated casein.
In the second part of this experiment, you will prove that the precipitated milk
product is a protein and compare a few simple properties of casein to free amino acids
and other proteins.
Biuret test. This is one of the most general tests for proteins. When a protein reacts with
copper (II) sulfate, a positive test is the formation of a copper complex that has a violet
color. This test works for any compound containing two or more peptide bonds.
Xanthoprotein test. This is a characteristic reaction of proteins that contain phenyl rings.
Concentrated nitric acid reacts with the phenyl ring to give a yellow-colored aromatic
nitro compound. Addition of alkali at this point will deepen the color to orange. The
yellow stains on the skin caused by nitric acid are the result of the xanthoprotein reaction
with tyrosine. Tryptophan results in a deeper orange color than tyrosine.
Cysteine test. Cysteine reacts with heavy metals to form a black precipitate. The
formation of such a precipitate indicates that at least two cysteine residues are present.
PROCEDURE:
Isolation of Casein
Weigh out 50 g of skim milk, recording the exact mass and volume. Add the milk to a
250-mL Erlenmeyer flask and heat the flask in a water bath. Stir the solution constantly.
Heat the bath and when the temperature has reached 40°C, remove the flask from the
water bath and add about 10 drops of glacial acetic acid while stirring.
Filter the mixture by vacuum filtration. Wash with 20 mL of 95% ethanol. Let the
vacuum run for 10 minutes to remove as much liquid as possible from the precipitated
casein.
Chemical analysis:
Each test should be done on each of the following six solutions:
a. 2% glycine
b. 2% gelatin
c. 2% albumin
d. 1% tyrosine
e. 1% cysteine
f. Casein prepared above from milk (1/4 of a spatula + 15 drops water, mixed well).
The casein is unlikely to fully redissolve as the precipitation caused the protein to
denature. Much of this denaturation is irreversible. The chemical tests below will
be unhindered by the presence of solid protein.
Biuret test:
Place 15 drops of each solution above in six different clean, labeled test tubes. To
each of the test tubes, add 7 drops of Biuret’s solution while swirling. Record your
results.
Xanthoprotein test:
This test should be performed in the hood. Place 15 drops of each of the solutions
above in six different clean, dry, labeled test tubes. To each test tube, add 10 drops of
concentrated HNO3 while swirling. Heat the test tubes carefully in a warm water bath.
Record your results.
Cysteine test:
Place 10 drops of each solution above in different clean, labeled test tubes. Add 5
drops of 3 M NaOH and 2 drops of 0.1 M lead nitrate. Heat the tubes for five minutes
and record your observations.
Protein denaturation:
Heat: Place 10 drops of each solution above in different clean, labeled test tubes.
Heat for 5 minutes and record your observations
Organics: Place 10 drops of each solution above in different clean, labeled test tubes.
Add six drops of methyl ethyl ketone, shake well and let sit for about 5 – 10 minutes.
Record your observations.
Acid: The xanthoprotein test can be used to see the impact of acid on protein folding.
All solutions should be disposed of the appropriate waste container in the hood.
