Aza Freya Lan A.

Tubato
BS BIOLOGY

PROTEIN HYDROLYSIS AND CHARACTERIZATION

EXPERIMENT NO.2
Introduction
To obtain information about the composition of a protein that has been isolated, one
method that is usually employed in the hydrolysis of the protein followed by the analysis of the
product.
In this experiment, the protein isolated in the first experiment will be subjected to acid or
alkaline hydrolysis. The acid/base hydrolysate together with the intact protein will then be
characterized using various of chemical tests.

A. Hydrolysis of Protein
MATERIALS
Protein isolate
8N H2SO4
16N H2SO4
Solid Ba(OH)2
Aluminum foil

50-mL Erlenmeyer flask

pH paper
Litmus paper
Autoclave

A.1 Acid Hydrolysis and Neutralization

1.
2.
3.
4.

50 mL Erlenmeyer flask add 5mL of 8N H2SO4 to of the protein isolate.

Label and plug with a piece of cotton. Cover with aluminum foil.
Autoclave for 15 psi for 5 hours.
Dilute the hydrolysate with 15 mL of distilled water then transfer the contents to a 250
mL beaker
5. Neutralize the hydrolysate by adding one spatulaful of Ba(oH)2. ten check the ph using
litmus paper.
6. If the hydrolysate is not yet neutral add saturated Ba(OH)2 solution dropwise.
7. filter off precipitate formed and wash with 2mL hot water twice.
8. if the volume of the filtrate is less than 15 mL, make up to 15 mL and then proceed to the
color reactions.--Autoclave for 15 psi for 5 hours.

Aza Freya Lan A. Tubato

BS BIOLOGY

B.2 Alkaline Hydrolysis and Neutralization

1. 50 mL Erlenmeyer flask add 5mL of boiling water and 3.0g of Ba(OH)2 to of protein
isolate.
2. Label and plug with a piece of cotton. Cover with aluminum foil.
3. Autoclave for 15 psi for 5 hours.
4. check pH. Confirm pH7 using pH paper.
5. filter off precipitate formed and wash with 2mL hot water twice.
6. if the volume of the filtrate is less than 15 mL, make up to 15 mL and then proceed to the
color reactions.
7. Dilute the hydrolysate with 15 mL of distilled water then transfer the contents to a 250
mL beaker
8. Neutralize the hydrolysate by adding 1.0mL of 16N H2SO4 dropwise. Check pH using
litmus paper
9. If the hydrolyzate is not yet neutral add 8N H2SO4 until pH7 is reached.

B. Color Reactions of intact Protein and Hydrolysis

MATERIALS
NaOH
0.01M CuSO4 solution
10% NaOH
0.02% naphtol solution
NaOBr (freshly prepared)
Mortar and pestle

0.01% ninhydrin solution

Conc. HNO3
Conc. NaOH
Hopkins-Cole reagent
Conc. H2SO4

PROCEDURE
Preparation of Protein Suspension:
1. Cut the dried casein into small pieces and palce in a mortar.
2. Add 30 mL of distilled water and grind until a fine protein suspension is obtained
Color Reactions
The First two tests should be done on a spot plot while the last three tests should be
done in small test tubes. Do the intact protein and hydrolysate samples side by side.
B.1 Biuret Test

Aza Freya Lan A. Tubato

BS BIOLOGY

Add 1 drop of 2.5M NaOH to 3 drops of the protein suspension and hydrolyzate samples.
Mix well

Add a drop or more of 0.01M CuSO4 solution. Mix well. Note the color produced.

B.2 Sakaguchi Test

1 drop of 10% NaOH and 0.02% naphthol solution to 5 drops of the protein suspension
and hydrolysate. Mix well

After about 3 mins add 1 drop of freshly prepared 2% NaOBr. Note the color produced.

B.3 Ninhydrin Test

To 10 drops of protein suspension add 1 mL of water. For hydrolysate, use 1 mL without

dilution with water.

Add 0.5 mL of 0.1% ninhydrin solution. Mix well

Heat in boiling water bath for 2-3 min. note the color produced.

B.4 Xanthoproteic Test

10 drops of protein suspension, add 1 mL of water, for acid/base hydrolysate use 1 mL

without tdilution with water.

Slowly add 3 drops of conc. HNO3. mix well. Note the color produced. Water bath for 1
min.

Cool the solution with flowing water then add conc. NaOH drop ny drop. Continue
adding until the solution is alkaline (test with litmus paper.)

B.5 Hopkins-Cole Test

2 mL of Hopkins-Cole reagent to 2 drops of protein suspension and hydrolysate.Mix well

Inclined the tube, add 2 mL of conc. H2SO4 slowly down the side of the tube until two
layers form.

Note the color formed at the interphase.

Aza Freya Lan A. Tubato

BS BIOLOGY

RESULTS:

Biuret test and Sakaguchi test

Xanthoproteic test

Ninhydrin test

Hopkins-Cole Test

Aza Freya Lan A. Tubato

BS BIOLOGY

Protein suspension

acid

base

Biuret Test

Violet complexation
reaction

Transparent with
cloudy particles

Remains the same

(transparent)

Sakaguchi Test

Turned into red to a

dark green

NaOH formed white

cloud turned dark
green

Turned into a dark

green

Ninhydrin Test

Cloudy ppt. Blue

Purple

Clear gray

Before heating:

dark blue

brown

dark blue

Xanthoproteic Test

Yellow

No change

No change

Before heating:

yellow orange

yellow orange

clear

Purple is the color of

the interphase

1st layer-pink

1st layer- cloudy

2nd- violet

2nd-white

3rd-blue

3rd- clear

After heating:

After heating:

Hopkins-Cole Test

4th layer- clear

GUIDE QUESTIONS:
1. Why is H2SO4/Ba(OH)2 used in the acid/alkaline hydrolysis of protein?

Aza Freya Lan A. Tubato

BS BIOLOGY

H2SO4/Ba(OH)2 in acid/alkaline neutralize the solution and balanced the

acidity and basic of the solution so you can get a neutral solution which is PH
7

2. What is the purpose of autoclaving?

The purpose of Autoclaving is a method to sterilize the solution.

3. Give the principle behind each chemical test done.

- In breaking down protein component into amino acid, we used protein hydrolysis.
We used some test to achieve it by using strong acid and strong base with an
enzyme to stimulate the natural hydrolic process.
Biuret Test- it is used to detect the presence of peptide bonds
Ninhydrin Test- it is used to detect the presence of amino acid in certain
substances
Sakaguchi Test- base catalyze condensation of naphthol with the guanidino group
of arginine
Xanthroproteic Test- it is the nitration of aromatic rings using electrophilic
aromatic substitution
Hopkin-cole Test- reduction of oxalic acid to glyoxilic acid and acid catalyze
condensation of 2 tryptophan with glyoxilic acid
4. Compare the results obtained with the acid and base hydrolyzates. Explain the
difference

Acid solution being positive for Sakaguchi, Ninhydrin, and Xanthoproteic

tests, the possible amino acid present in the composition of the acid
hydrolyzate consist of amino acid arginine, a free a-amino group, and an
amino group with phenyl ring. In Basic solution being positive for the
Ninhydrin, Xanthoproteic, and Hopkins-Cole tests, the possible amino acid
present in the composition of the basic hydrolyzate consist of a free a-amino
group, an amino group with a phenyl ring, and amino group tryptophan.
Therefore unique amino acids exist, and because of their uniqueness to one
another, a series of tests can be performed to detect specific amino acids

Aza Freya Lan A. Tubato

BS BIOLOGY

Experiment no.1
Isolation of casein from milk

Proteins are polymers of amino acids linked by peptide bonds. They are relatively easy to
separate from other biomolecules such as nucleic acids, lipids, and carbohydrates. However, they
are more difficult to separate from each other because they are similar in such structure and
properties. Thus, small differences in properties, such as size, charge and ability to bind specific
groups are used to separate them.
Milk from cows has an average composition of 87.1% water, 3.4% protein, 3.9% fats, 4.9%
carbohydrates and 0.7% minerals. There are three kinds of proteins in milk: casein, lactalmumins,
and lactoglobulins. All three are globular proteins and are considered complete proteins because
they contain all the amino acids necessary for building blood and tissue. These proteins can
sustain life and provide normal growth. Casein is the main protein in milk and exists as the
calcium salt, calcium caseinate. It has an isoelectric point of pH 4.6.

In this experiment, casein will be isolated from non-fat milk by isolectric precipitation.

MATERIALS

Powdered non-fat milk (5g)

10% Acetic Acid

EQUIPMENT

pH meter
Suction filtration set-up

Aza Freya Lan A. Tubato

BS BIOLOGY

PROCEDURE

1. In a 100 mL beaker, dissolve 50 of powdered non-fat dry milk in 20 mL warm distilled

water.
2. Heat the solution to 550 C on a hot plate
3. Remove the beaker from the hot plate.
4. Take note of the initial pH of the milk solution and then add dropwise a solution of acetic
acid while stirring with a stirring rod.
5. Continue adding the acid solution until the pH reaches 4.6 take note of the volume of acid
used.
6. Let stand until a large amorphorus mass is formed.
7. Filter off the precipitate by suction filtration or gravity filtration.
8. Dry the isolated casein between filter papers and then weigh the casein. Determine %
yield.
9. Divide the isolated casein into two portions. Use 1 portion for acid/base hydrolysis. Store
the other portion in the refrigerator( tobe characterized later susing various chemical
tests)

Aza Freya Lan A. Tubato

BS BIOLOGY

RESULTS:

Aza Freya Lan A. Tubato

BS BIOLOGY

GUIDE QUESTIONS:

1.Why is non-fat milk, instead of whole milk, used in experiment?

we used non-fat milk instead of whole milk in this experiment as casein maybe extracted
both from whole milk and non-fat milk. because the disadvantage of whole milk is that it
contains relatively large concentration/amount of fat rather than non-fat/fat mat. This excess
fat could or will be included in the separation of casein but what we want is pure casein.
2.Why is the milk solution heated at 55 degree C, but not higher, on a hot plate?

We heated the milk to 550 C on a hot plate. The temperature while heating should not exceed
55oC. The milk is not heated above 550 C because then it would curdle. The milk would
become thicker because the overheating will remove the water content. Heating up to 550 C is
just right for denaturing the milk.
3. Explain the principle behind the isolation of casein from milk.

The principle behind isolation of casein from milk is isoelectric precipitation. Casein has an
isoelectric pH of 4.6 therefore insoluble in solutions with ph lower than 4.6. The ph of milk is
about 6.6 which gives casein a negative charge and makes it soluble. Once you add an acid to
the solution, the negative charge of casein becomes neutral, precipitating the casein.