Analysis and Denaturation of Proteins

Experiment no.4

Theory
The cell is composed mainly of proteins, and their repeating unit, the amino acid. Protein functions as biological
catalysts or enzymes, transporters of oxygen, and hormones.

There are four levels of protein structure: the primary, secondary, tertiary and quaternary levels. The primary
structure is composed of single covalently-bonded amino acids. There are two types of secondary structure, the α-helix
and the β-sheets. The alpha helix structure contains one strand of amino acid chain that is bonded by intramolecular
hydrogen bonds. Two chains that are linked by hydrogen bonds describe the beta-sheet structure. The tertiary structure
of the protein refers to the combination of either pure helix or of pure beta or a combination of both. The stabilizing
interactions in the tertiary level are (a.) salt linkages, (b) hydrogen bonds, (c) disulfide linkages, and (d) hydrophobic
interactions.

Denaturation of Proteins.

Denaturation is the unfolding of the complex secondary, tertiary and quaternary structure of proteins. Heat, strong
acids and organic solvents (e.g., alcohol) can denature proteins. Heat causes the atoms within the protein molecule to
vibrate more rapidly, causing the hydrogen bonds and hydrophobic interactions to break. Strong acids split salt linkages
by ionizing the carboxylic group,
and alc
ohol denatures the protein by disrupting the hydrogen bonds.

Heavy metals ions like silver, lead and mercury also denature proteins by combining the free carboxylate anions of
the acidic amino acid with the metal, causing precipitation. This is the rationale in the use of proteins (e.g., egg white) as
antidote for heavy metal poisoning.

Organic acids, like picric acid and tannic, are used to precipitate the alkaloids giving rise to the name “alkaloidal
reagents”. The anion of the acid will react with the protonated amino groups of protein, causing disruption of the salt
linkages. Leather is manufactured by coating animal skins with tannic acid, hence the process is known as tanning.

Color Tests of Proteins

Amino acids can be identified by the R-groups attached to the α-carbon that reacts with specific chemicals.
However, since proteins contain various amino acids, one test will not be enough to identify the amino acids. It would be
best to perform numerous tests before concluding the components of a protein.

1. Biuret Test
This test will give a positive result for compounds that contain 2 or more peptide linkages. It will give a
distinctive purple color which is probably due to the formation of a complex by cupric ions with the amino groups
called Biuret. Hence, dipeptides and amino acids like serine and threonine do not give positive results with this
test.

2. Ninhydrin Test
When amino acids are sprayed with ninhydrin, it will give a blue to violet colored result. The presence of
amino group including those found in amines and ammonia will also give the same result except for praline and
hydroxyproline that will give a yellow colored result.

3. Xanthoproteic Test
Nitration of amino acids that contain benzene ring will yield the product nitrobenzene that will give a
yellow to orange coloration. Tryptophan, tyrosine and phenylalanine will produce nitrobenzene when treated with
concentrated nitric acid. Collagen and gelatin do not give a positive reaction.

4. Millon’s Test
The presence of phenol group in amino acid like tyrosine is nitrated by a solution of mercuric and
mercurous nitrates in concentrated nitric acid. A white precipitate will start to form, turning brick red on
prolonged hating due to the formation of a mercury complex of nitrophenyl derivatives. Addition of NaNO 2 turns
the precipitate to darker pink or red.

5. Hopkin’s Cole Test

 The aldehyde present in the reagent will cause the formation of a blue or violet colored ring due to the
formation of a complex between the reagent and the indole ring of tryptophan. Gelatin and collagen fail to give a
positive result with this test indicating the absence of tryptophan in these proteins.

6. Sakaguchi Test
The combination of alpha-naphtol and sodium hypobromite or hypochlorite will react with the guanido
group giving a red to orange colored complex. This test is used to identify the presence of arginine. This is an
extremely sensitive test and may be used as a general test for proteins, because all known proteins contain sufficient
arginine.

7. Lead Acetate Test (Test for Sulfur)

This test is specific for sulfur-containing amino acids like cysteine and methionine. The sulfahydryl or
disulfide groups are converted to inorganic sulfide, Na2S, in strongly alkaline solution. This will react further with
lead acetate to form a brownish-black precipitate of lead sulfide (PbS).

Objective
To be able to identify the different amino acids in a protein sample.

Materials
Test tubes Tyrosine
Test tube holder Millon’s reagent
Test tube holder 0.1% NaNO2
Test tube rack Hopkin’s Cole reagent
dropper conc. H2SO4
pipette 10% NaOH
aspirator 5% lead acetate solution
spatula ammonia water
stirring rod 0.2% urea
Bunsen burner 95% ethanol
Tripod 2% mercuric chloride
Wire gauze 2% lead acetate
Watch glass 2% silver nitrate
Water bath Picric acid
Graduated cylinder Tannic acid
Cork egg albumin (from fresh egg)
5% albumin
5% gelatin
conc. HNO3
conc. NH4OH
5% phenol

Procedures

I. Color Tests for Amino Acids and Proteins

A. Xanthoproteic Test
Place 1 ml each of 5% albumin solution, 5% gelatin solution and tyrosine in separately-labeled test tubes. Add 5
drops of conc. HNO3 into each tube. Notice the formation of white precipitate. Mix it thoroughly, heat in a water bath, and
note the change in color. Allow it to cool and add a few drops of conc. NH 4OH. Note the intensity of the color.

Xanthoproteic Test Observations

Albumin
Gelatin
Tyrosine

B. Millon’s Test
Place 1 ml each 5%solution of albumin, tyrosine and 5% phenol solution in separately-labeled test tubes. Add 4
drops of Millon’s reagent into each test tube. Heat in boiling water bath for 10 minutes, then allow it to cool by placing the
tube in running tap water. Then add 4 drops of freshly prepared 0.1% NaNO 2 and warm gently. Note any change in the
color of the precipitate.

MIllons’ Test Observations

 Albumin
Tyrosine
Phenol

C. Hopkin’s Cole Test

Place 1 ml each of tyrosine, albumin solution and 5% gelatin solution in separately-labeled test tubes. Add 4 drops
of Hopkin’s Cole reagent. Incline the test tube in the test tube rack. Carefully, allow 2 ml of conc, sulfuric acid to slide
down the side of the inclined tube so that it will form a layer below the protein solution. Take note of the color of the
junction of the two liquids.

Hopkin’s Cole Test Observations

Albumin
Gelatin
Tyrosine

D. Lead Acetate Test

Place 1 ml of 5%albumin solution, 5% gelatin solution in separately labeled test tubes. Add 5 drops of 10% NaOH,
and 3 drops of 5% lead acetate solution into each test tube. Shake and heat in boiling water bath. Describe the color of the
precipitate formed.

Lead Acetate Test Observations

Albumin
Gelatin

E. Biuret Test
Place 2 ml each of 5% albumin and urea in separately-labeled test tubes. Add 1 ml of 10% NaOH solution and
about 5 drops of CuSO4 solution. Observe the color that develops.

Biuret Test Observations

5% Albumin
Urea

II. Protein Denaturation

A. Coagulation
1. By Heat
Label 2 test tubs as 1 and 2. To both tubes, place a small amount of egg albumin solution. To tube 1, add 3 ml
distilled water and heat the test tube in a boiling water bath for 10 minutes stirring. Allow it to cool. Add 3 ml distilled
water to test tube 2 and stir. Filter both tubes separately, then test both filtrates using Biuret reagent. Compare the results
and record your result below.

Observations
Test tube 1
Test tube 2

2. Inorganic Acids
Label two test tubes as 1 and 2, and add 3 ml of 5% albumin into each tube. Test tube 1, add concentrated HCl drop
by drop, shaking after each addition. Record the number of drops of the acid added until a precipitate is formed. Then add
an excess amount of HCl and take note whether it will increase or dissolve the precipitate formed. Repeat the same
procedure in test tube 2, but this time use concentrated H2SO4.

Observations
HCl
H2SO4

3. Alcohol
Place 1 ml each of 5 % solutions of albumin and gelatin in separately-labeled test tubes. Add 5 ml of 95% ethanol
in each test tube, mix and note the formation of precipitate.

Observations
 Albumin
Gelatin

B. Precipitation

1. By Heavy Metal Salts

In three test tubes, place 3 ml of egg albumin solution. To test tube 1, add 5 drops of lead acetate solution. Add
excess drop of lead acetate solution and note whether the precipitate is increased or dissolved. Repeat the procedure by
adding 5 drops of silver nitrate to test tube 2 and 5 drops of mercuric chloride to the third test tube. Describe the results.

Heavy metal salts Observations

Lead acetate
Silver nitrate
Mercuric chloride

2. By Alkaloidal Reagents
Place 3 ml of egg albumin solution in two test tubes. In test tube 1, add 2 ml of tannic acid solution, and in test tube
2, add 2 ml of picric acid solution. Describe the protein solution in each of the tubes after the addition of alkaloidal reagents.

Alkaloidal Reagent Observations

Picric acid
Tannic acid
 CAGAYAN DE ORO COLLEGE-PHINMA

Biochemistry Laboratory Final Report

Name: ___________________ Rating: _____________

Course/Year/ Section: ______ Date Performed: ____________
Group no. _______________ Date Submitted: ____________

Experiment no. _____

Title: ___________________________________________________________
________________________________________________________________

Objective: _______________________________________________________
________________________________________________________________
________________________________________________________________

Materials: ______________ ________________

______________ ________________
______________ ________________
______________ ________________

I. Color Tests for Amino Acids and Proteins

A. Xanthoproteic Test

Xanthoproteic Test Observations

Albumin
Gelatin
Tyrosine

B. Millon’s Test

MIllons’ Test Observations

Albumin
Tyrosine
Phenol

C. Hopkin’s Cole Test

Hopkin’s Cole Test Observations

Albumin
Gelatin
Tyrosine

D. Lead Acetate Test

Lead Acetate Test Observations

Albumin
Gelatin

E. Biuret Test

Biuret Test Observations

5% Albumin
Urea

II. Protein Denaturation

 A. Coagulation
1.By Heat

Observations
Test tube 1
Test tube 2

2. Inorganic Acids

Observations
HCl
H2SO4

3. Alcohol

Observations
Albumin
Gelatin

B. Precipitation
1. By Heavy Metal Salts

Heavy metal salts Observations

Lead acetate
Silver nitrate
Mercuric chloride

2. By Alkaloidal Reagents

Alkaloidal Reagent Observations

Picric acid
Tannic acid

Questions
1. Surgical instruments are sterilized by heating them, and alcohol is used as disinfectant in cleansing the skin prior
to an injection. Why are these methods successful in killing harmful microorganisms?
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2. Explain why egg whites and milk are used as antidotes for heavy metal poisoning.
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3. Explain why picric acid and tannic acids are sued in the treatment of burns.
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4. What is the colored precipitate obtained in the sulfur test (lead acetate test)?
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Conclusions
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