Name: Nabor, Elgie L.____________________ Yr. & Sec.

: BSN 1-YB-24________ Score: __________

Date: April 1, 2023_____________ Instructor: Ms. Jenny Lyn Operario_________________________

EXPERIMENT 5_________________________________________________
 ANALYSIS AND DENATURATION OF PROTEINS

THEORY
The cell is composed mainly of proteins, and their repeating unit, the amino acid. Proteins
function 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 ß-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 alcohol denatures the protein by disrupting the hydrogen bonds.

Heavy metals ion 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 acid, are used to precipitate the alkaloids giving
rise to the name “alkaloid 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 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 ( 2 mL 10% NaOH + 5 drops CuSO4)
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 proline 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.
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 heating due to the formation of a
mercury complex of nitro phenyl derivatives. Addition of NaNO2 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
PROCEDURES
I. Color Tests for Amino Acids and Proteins
A. Xanthoproteic Test
Place 1 mL each of 5% albumin solution, 5% gelatin solution, tyrosine and
phenylalanine in separately labeled test tubes. Add five 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. NH4OH.
Note the intensity of the color.

Xanthoproteic Test Observations

Albumin Before heated, the color of the solution is yellowish. After

heating it, the color turned into bright yellow and it is semi-
solid. Upon shaking it, the color turned into foggy yellow and
it became semi-liquid.

Gelatin Before heated, the color of the solution is yellowish. After

heating and shaking it, the color is still the same. However,
the characteristics of the solution is dilute.

Tyrosine Before heating it, the color of the solution is brown. After
heating it, the color turned into light yellow. Upon shaking it,
the color became lighter yellow.

Phenylalanine Before heating it, the color of the solution is yellowish. After
heating and shaking it, the color is still the same. There’s no
visible result.

B. Millon’s Test
Place 1 mL of 5% albumin solution, 5% gelatin solution, tyrosine and phenol
solution in separately labeled test tubes. Add four drops of Millon’s reagent into each
test tube. Heat in boiling water bath for 10 minutes, and then allow it to cool by placing
the tube in running tap water. Then, add four drops of freshly prepared 0.1% NaNO2
and warm gently. Note any change in the color of the precipitate.

Millon’s Test Observations

Albumin The albumin solution is color white when heated; After

 heating and adding NaNO2, the color turned foggy white.
Then when we shake it, the color turned pale pink and
there’s white curd at the top.

Tyrosine The tyrosine solution is dark yellow when heated; After

heating and adding NaNO2, it turned into clear brown. Then
when we shake it, the color turned reddish dark brown and
there’s a small particles floating.

Phenylalanine The phenylalanine is clear light yellow when heated; After

heating and adding NaNO2, it turned into clear white. Upon
shaking it, there’s no changing in color. It still clear white.

Phenol The phenol is color red when heated; After heating and
adding NaNO2, reddish black precipitate formed and the
color of the top solution is clear yellow. Upon shaking it, the
solutions turns into light brown but there was still reddish
black precipitate.

C. Hopkin’s Cole Test

Place 1 mL of each of tryptophan, tyrosine, 5% albumin solution, and 5% gelation
solution in separately labeled test tubes. Add four 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 lipids.

Hopkin’s Cole Test Observations

Albumin In my observation, the color of the solution is clear yellowish

white. And, at the top of the solution there is a white
bubbles or curd floating in the solution.

Gelatin Upon observation, I saw three colors in test tube. At the top,
the color is clear white. At the bottom, the color is brownish
clear white. And, a yellowish black ring is formed at the
junction between the 2 layers.

Tyrosine The color of the solution is reddish brown. The bottom part is
 clear white and there is a small particles floating in the
solution. After a few minutes of observing, I observed that
there is no changing in color. It is still the same.

Trytophan Upon observing, there is white bubble at the top of the

solution. The bottom is clear white. The middle of the
solution is color yellowish with a purple ring formed at the
junction between the 2 layers. Also, I observed that there is
one black particle floating in the solution.

D. Lead Acetate Test

Place 1 mL of each 5% albumin solution and 5% gelatin solution in separately labeled
test tubes. Add five drops of 10% NaOH, and three drops of 5% lead acetate
solution into each test tube. Shake and hear in boiling water bath. Describe the Color
of the precipitate formed.

Lead Acetate Test Observations

Albumin After heating and shaking it, the solutions became black and
white precipitate formed.

Gelatin The gelatin’s color is white when heated; after heating and
shaking it, the color turned yellow and there’s cloudy white
particles or curd under it.

E. Ninhydrin Test
Place 2 mL each of 5% solution of albumin, ammonia water, and 0.2% urea in separately
labeled test tubes. Add 1 mL of 0.1% freshly prepared ninhydrin reagent into each
test tube. Using a Cork, cover the test tube tightly and boil over a bath. Observe the
color produced.

Ninhydrin Test Observations

Albumin Before heated it, the color of the solution is less foggy white.
After heating it, the color of the solution produced more
foggy white.

Ammonia water Before heated it, the color of the solution is clear white.
After heating it, the color became yellowish white in color.
 Urea Before heated it, the color of the solution is clear white.
After heating it, the color is still clear white. There’s no
visible result.

F. Biuret Test
Place 2 mL each of 5% albumin, urea and alanine in separately labeled test tubes. Add 1
mL of 10% NaOH solution and about five drops of CuSO4 solution. Observe the color
that develops.

Biuret Test Observations

5% albumin The color blue gradually changed into violet and there is a
white color mixing it.

Urea The color has gradually changed. From blue, it became dark
green.

Alanine After waiting for a few minutes, the blue solution slowly
changes color into dark violet color.

II. Protein Denaturation

A. Coagulation
1. Heat
Label two test tubes 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 with constant stirring. Allow 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 findings.

Observations

Test tube 1 Before heated, the color of the solution is blurred white.
After heating, filtering and adding reagent the solution
became light blue and there is some particles.

Test tube 2 Before filtering and adding reagent, the color of the solution
is dirty white. After filtering and adding reagent the solution
 became light blue. However, unlike to test tube 1. The test
tube 2 has no particles.

2. Inorganic acids
Label two test tubes as 1 and 2, and add 3 mL of 5% albumin into each tube. To 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

HCI After 15 drops of concentrated HCI, it formed a white

precipitation. The top of the solution formed a curd and the
bottom became dirty white.

H2SO4 After 8 drops of concentrated H2SO4, it precipitates. The

color of the solution is brown. After mixing it, it became dark
violet. The top of the solution formed a curd. Then, there
are small particles that settle at the bottom

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

Observations

Albumin After adding ethanol and mixing it, there is white precipitate.
The bottom is foggy white and the top is blurred white.

Gelatin After adding and mixing it, there is also white precipitate.
The solution became blurred white and there is small
particles floating it.

Peptone After adding and mixing it, the solution became clear white.
However, there are small particles at the bottom.
B. Precipitation
1. Heavy Metal Salts
In three tubes, place 3 mL of egg albumin solution. To test tube 1, add five 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 five drops of
silver nitrate to test tube 2 and five drops of mercuric chloride to test tube 3.
Describe the results.

Heavy Metal Salts Observation

Lead acetate At first, the solution is formed a blurred clear curd on top.
After 5 drops of lead acetate it formed a blurred white like
cloudy on the back and side. And the bottom also formed
curd. After excess drops the precipitation increased.

Silver nitrate Like lead acetate, the solution also formed a blurred clear
curd on top. After 5 drops of silver nitrate, a string like
cloudy white formed. After excess drops, the precipitation
increased.

Mercuric chloride Mercuric chloride also formed a blurred clear curd on top at
first. After 5 drops of mercuric chloride it formed a curd on
top and the curd does not reach at the bottom. After excess
drops, the precipitation decreased.

2. 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 Reagents Observation

Tannic Acid At first the color of the solution are blurred white. After
adding same solutions the color became dirty brown. Then
after adding reagents, it became yellowish brown and curd at
the bottom formed.

Picric Acid Like tannic acid, the color of the picric acid is blurred white.
After adding same solutions the color became bright yellow.
Then after adding reagents, it is still bright yellow but curd at
 the bottom formed.

QUESTIONS
1. Surgical instruments are sterilized by heating them, while alcohol is used as a
disinfectant in cleansing the skin prior to injection. Why are these methods successful
in killing harmful microorganisms?
- Heat for sterilization and alcohol for disinfection are both successful methods of
killing harmful microorganisms because heat (the most common method of physical
sterilization) can kill microorganisms by altering their membranes and denaturing
proteins. As the temperature rises, the weakest bonds that keep protein structures
together start to break. Also, high temperatures can dehydrate the microorganisms.
On the other hand, alcohol can kill the microorganism through denaturation and
coagulation of proteins. In addition, it is a great way to cleanse the skin before
injection to kill the pathogens present in the skin because we cannot use heat to
sterilize our skin because this method can harm us physically.
2. Explain why egg whites and milk are used as antidotes for heavy metal poisoning.
- Egg whites and milk are used as antidotes for heavy metal poisoning because they
contain proteins that can bind to heavy metal ions, such as lead or mercury. The
proteins of egg whites and milk can coat the mucus membrane liner of the
heavy metal which can hinder the absorption of poison in the body.
3. Explain why picric acid and tannic acids are used in the treatment of burns.
- Picric acid and tannic acids are used in the treatment of burns because they contain
astringent and antiseptic. Astringents allow the tissues and cells of the skin to
tighten, which can aid in reducing swelling and inflammation. On the other hand, by
eradicating or preventing the growth of bacteria, antiseptics aid in the prevention of
infection. Furthermore, both picric acid and tannic acid have the ability to denature
proteins, which can aid in forming a barrier of defense around the burn to stop
further damage and promote healing.
4. What is the colored precipitate obtained in the sulfur test (lead acetate test)?
- In our experiment, the colored precipitated obtained or formed in the sulfur test
(lead acetate test) is color black.