Saint Mary’s University

School of Health and Natural Sciences

BAYOMBONG, NUEVA VIZCAYA 3700
Nursing Department
Laboratory No. 7: Analysis and Denaturation of Proteins

CARBONEL, Kyla Mae M.

BSN 1A

ABSTRACT

Proteins are the nutrients that we could get in eating meat. That could easily detect by a urine test
that being done in a laboratory. Proteins that we need in our diet depend on our weight, age and
health. But in this activity we are going to test how the proteins react on different products or
different substances, and on how protein becomes an antidote for heavy metal poisoning.
Denaturation is the unfolding of the complex secondary, tertiary and quaternary structure of
proteins. Heat, strong acids and organic solvents can denature proteins and that is used in doing
this activity.

Keywords: Antidote, Complex, Heat, Nutrients, Strong Acids

I. INTRODUCTION

Protein denaturation is the net effect of alterations in the biological, chemical, and physical
properties of the protein by mild disruption of its structure. When blood samples are taken for
protein analysis, it is important that they are handled correctly so that no artifacts are introduced
that could affect the investigation and its interpretation. If the protein is allowed to even partially
degrade, the assay will not be accurate. Therefore, it is essential that denaturation is avoided. The
ability of plasma proteins to resist denaturation in a blood sample taken for diagnostic analysis
varies between proteins; consequently, the sample should be handled according to the analysis
required. Fortunately, most major plasma proteins are relatively resistant to denaturation and can
be assayed in samples that have been handled carefully and have been kept away from elevated
temperatures. However, separation of plasma or serum from the blood cells
by centrifugation should be performed as early as possible. Thereafter, many proteins are stable at
4°C for several days and at –20°C for much longer (months to years). Some proteins are less stable,
with enzymes being particularly susceptible to loss of activity with time, while the stability of the
peptide hormone ACTH is so low that samples should be snap-frozen immediately to preserve the
intact peptide.

BIOCHEMISTRY CARBONELBSN1-A
 For identification and quantification of serum protein, the protein component in serum must
either be separated or individual proteins must be measured independently. The primary separation
of the proteins in serum is between albumin and the globulins. Albumin is a water-
soluble, globular protein that is usually identifiable as a single discrete molecule. The globulins
are also globular proteins, but many of them, in contrast to albumin, precipitate in pure water and
require salts to maintain their solubility. The globulins are a mix of proteins of various types, which
migrate in groups in an electric field (electrophoresis) as families of proteins identified as α-, β-,
or γ-globulins. The nomenclature of the globulin fractions is based on their location during
separation by electrophoresis. Albumin has the most rapid migration of the major proteins (in some
species it is preceded by prealbumin), followed by the α-globulin, β-globulin, and γ-
globulin fractions, respectively. The γ-globulins are largely composed of immunoglobulins, the
antibodies that bind to invading pathogens or other foreign matter. In contrast, the α- and β-
globulin fractions contain a great variety of different proteins (P.David Eckersall, 2008).

II. OBJECTIVES

To be able to identify amino acids in a protein

III. MATERIALS

 5% albumin
 5% Gelatin
 Concentrated HNO3
 Concentrated H2SO4
 5% Phenol
 Alanine
 Tyrosine
 Tryptophan
 Phenylalanine
 Millon’s Reagent
 0.1% NaNO2
 Hopkin’s Cole Reagent
 10% NaOH
 5% lead acetate solution
 Peptone
 0.1% ninhydrin Reagent
 Ammonia Water
 0.2% Urea
 95% ethanol

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  2% mercuric Chloride
 2% lead Acetate
 2% Silver Nitrate
 Picric Acid
 Tannic Acid
 Concentrated HCl
 Egg Albumin

IV. PROCEDURES

A. Color Tests for Amino Acids and Proteins

1) Xanthroproteic Test
a) Place 1 mL each of 5% albumin solution, 5% gelatin solution, tyrosine, and phenylalanine
in separately labeled test tubes. Add 5 drops of concentrated HNO3 into each test tube.
Notice the formation of white precipitate. Mix it thoroughly, heat in water bath, and note
the change in color. Allow it to cool and add a few drops of concentrated NH4OH. Note
the intensity of the color.
b) Record all results.
2) Millon’s Test
a) Place 1 mL each of 5% albumin solution, tyrosine, phenylalanine, and 5% phenol in
separately labeled test tubes. Add 4 drops of Millon’s Reagent into each test tube. Heat in
boiling bath for 20 minutes, then allow to cool by placing the tube in running tap water.
Then add 4 drops of freshly prepared 0.1% NaNO2 and warm gently. Note any change in
color of the precipitate.
b) Record your results.
3) Hopkin’s Cole Test
a) Place 1 mL of tryptophan, tyrosine, 5% 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 1mL concentrated H2SO4 to slide down the side of
the tube so that it will form a layer below the protein solution. Take note of the color of the
junction of the two liquids.
b) Record the results.
4) Lead Acetate Test
a) Place 1mL each of 5% albumin solution, 5% gelatin solution in separately labeled test
tubes. Add 5 drops of 10% NaOH, add 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.
Record results.

BIOCHEMISTRY CARBONELBSN1-A
5) Ninhydrin Test
a) Place 2 mL of 5% solution of albumin, ammonia water, 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 water bath. Observe the color produced.
6) Bluret Test
a) Place 2 mL each of 5% albumin, urea and alanine in separately-labeled test tubes. Add 1
ml of 10% NaOH solution and about 5 drops of CuSO4 solution. Observe the color that
develops.
B. Protein Denaturation
1) By Heat
a) Label 2 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 it cool. Add 3 mL distilled water to test tube 2 and
stir. Filter both tubes separately, then test both filtrates using Bluret Reagent. Compare the
results and record.
b) Record your results.
2) Inorganic Acids
a) Label 2 test tubes as 1 and 2, add 3 mL 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 wheter it will increase or dissolve the precipitate formed. Repeat the same procedure
in test tube 2, but this time use concentrated H2SO4.
b) Record your observation.
3) Alcohol
a) Place 1 mL of each 5% solution of albumin, gelatin and peptone in separately-labeled
test tubes. Add 5 mL of 95% ethanol in each test tube, mix and note formation of
precipitate.
C. Precipitation
1) By Heavy Metal Salts
a) 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 whetehr the
precipitate is increased or dissolved. Repeat the procedure by adding 5 drops of silver
nitrate to test tube 2 and 3 of mercuric chloride to the third test tube. Describe the
results.
2) By Alkaloidal Reagents
a) Place 3 mL of egg albumin solution in two test tubes. In test tube 1 add 2 mL of tachnic
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.

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V. 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?
2. Explain why egg whites and milk are used as antidotes for heavy metal poisoning.
3. Explain why picric acid and tahnic acids are used in the treatment of burns.
4. What is the colored precipitate obtained in the sulfur test (lead acetate)?

VI. RESULTS AND DISCUSSION

Table 1: Results of the substance that is tested in the xanthoproteic Test

Substance tested Description of visible results Intensity of color

Albumin Precipitation occurs (white-
yellow)
Gelatin Clear
Tyrosine Yellow that looks like urine
Phenylalanine Small drops or portion of yellow
The table contains the results and how the intensity of the color is being tested

Table 2: Substances that reacts to Millons Reagent

Substance Tested Results

Albumin Cloudy white
Tyrosine Cloudy white at the bottom
Phenylalanine White
Phenol Brownish Red
In this table it contains the result of the protein solutions after the Millons Reagent is added

Table 3: Color of the substances after Hopkins cole reagents is added

Substance Tested Results

Albumin Brown spots at the base and cloudy at the
upper part
Gelatin Occurence of brown ring at the middle
Tyrosine Green color at the base of test tube
Trytophan Yellow base while the other half is clear

BIOCHEMISTRY CARBONELBSN1-A
 The table contains the result after the Hopkins Cole reagent is added and also the H2SO4 to
the proteins solution

Table 4: Lead Acetate test for the 2 substances

Substance Tested Results

Albumin All black
Gelatin Clear
It contains the color of the precipitation that is formed when it is heated and shaked after
the lead acetate is added

Table 5: Color produced by ninhydrin reagent

Substance Tested Results

Albumin On the upper light blue while in the middle
blue and at the bottom clear
Ammonia Water Looks like red wine
Urea Clear with bubbles
The color produced then the ninhydrin is added and boil over the water bath is containing
this test tube

Table 6: Color develops from NaOH and CuSO4

Substance Tested Results

Albumin Cloudy and violet at the top
Alanine Blue particles compact
Urea Blue particles are spread
This table has the results of the color produced after adding a NaOH and CuSO4 solution
to the protein solution

Table 7: Comparing test tube when it is boiled and not

Results
Test tube 1 Clearer liquid than test tube 2
Test tube 2 cloudy

BIOCHEMISTRY CARBONELBSN1-A
 While in this table has the results of comparing two test tubes with same solution but other
is put on a boiling water and the other is not.

Table 8: Testing substances using inorganic substance or acids

Results and observation

HCl Denser cloudy particles at the bottom than
the top
H2SO4 Less denser cloudy particle on the bottom
Both tubes are added by an inorganic acids but different acid until the precipitate is formed
and it will dissolved or increased the precipitate.

Table 9: Proteins that reacts to alcohol

Results and observation

Albumin It becomes cloudy particles and at the
bottom there’s a precipitation
Gelatin No reaction
Peptone It becomes light yellow
This table has the results of adding an alcohol to the protein solution and on the reaction

Table 10: Heavy Metal Salts are tested or used

Heavy Metals Tested Results and Observation

Lead Acetate Dirty white with white particles at the
bottom
Silver Nitrate Cloudy white with mix tiny and large
particles
Mercuric Chloride White in color with a little bit of color
yellow with some tiny particles at the
bottom
All test tube are added by the same protein solution but differs on the metal that added

Table 11: Alkaloidal Reagents are used or being tested in the protein solution

Alkaloid Reagent Results and Observation

Picric Acid It becomes color yellow
Tahnic Acid It becomes reddish brown
Same in the table 11 same protein solution is added the egg albumin but different alkaloidal
reagent is used

BIOCHEMISTRY CARBONELBSN1-A
 Normally microbes can't endure temperatures higher than 45 degrees Celsius, which is the
reason careful instruments are sanitized at the temperature of 100 degrees Celsius, just on the off
chance that a portion of the microscopic organisms figure out how to endure. Alcohol likewise
clears your skin of microorganisms by dissolving their cell layers leaving them presented to the
environment and their up and coming passing (Greenleafable,N.D).
Heavy metals salts act to denature proteins in much the same manner as acids and bases,
heavy metal salts usually contain Hg+2, Pb+2, Ag +2, TI+1, Cd+2 and other metals with high atomic
weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal
salt with a protein usually leads to an insoluble metal protein salt. This reaction is used for its
disinfectants properties in external applications. For example AgNO3 is used to prevent gonorrhea
infections in the eyes of new born infants. Silver nitrate is also used in the treatment of nose and
throat infections, as well as to cauterize wounds. Mercury salts administered as Mercurochrome
or Merthiolate have similar properties in preventing infections in wounds. This same reaction is
used in reverse in cases of acute heavy metal poisoning. In such a situation, a person may have
swallowed a significant quantity of a heavy salt. As an antidote, a protein such as milk or egg
whites may be administered to precipitate the poisonous salt. Then an emetic is given to induce
vomiting so that the precipitated metal protein is discharged from the body. (Namrata Chhabra,
2011).
In the First World War the treatment of burns consisted in the application of various
antiseptic dressings. Picric acid in a 1 per cent aqueous solution was commonly used. The
treatment by picric acid carried with it some danger of absorption when used over large raw
surfaces, but it was generally a very satisfactory form of treatment. It controled pain and rapidly
promoted healing. The use of tannic acid was a distinct advance in the treatment of cutaneous
burns and found the care of patients relatively simple.Beneficial effects: rapid alleviation of pain,
lessened infection, reduced local loss of body fluids, prosperous epithelization, relative absence of
contractures, and diminished scar-tissue formation.Most important of all, good survival statistics
were reported in several studies. In a group of 114 patients treated with tannic acid, Beekman
observed a mortality rate of 14.9 percent, which was considerably lower than the death rate of 27.8
percent in the 320 patients treated otherwise. He also showed that the number of patients dying
from the second to the tenth day, the period of toxemia, was strongly decreased in the tannic acid-
treated group. (.answers.com)

The product of this reaction is black lead (II) sulfide. It forms upon the reaction between lead
(II) acetate and the sulfur in the egg. C. dilute sodium hydroxide in test tube: NaOH ---> Na+ +
OH- addition of dry egg albumin: 2Na+ + S ---> Na2S ---> 2Na+ + S2- addition of lead acetate:
S2- + Pb2+ ---> PbS Because of their peptide structure and the presence of different amino acid
groups in their molecules proteins react with a variety of agents to form colored products. Try each
of the protein tests on the following solutions of proteins and protein derivatives. 2.0% casein
.Dissolve casein in weak alkali and neutralize using indicator paper. 2.0% egg albumin. Dissolve
egg albumin in water without heating and with a minimum offrothing. 1.0% gelatin. Dissolve
gelatin in water with the aid of heat. 0.5% tyrosine. Suspend tyrosine in water and bring into
solution by adding HCl. 0.1% tryptophan. Dissolve in water with the aid of heat. 0.5% cystine.

BIOCHEMISTRY CARBONELBSN1-A
 Suspend in water and bring into solution by adding a little conc. HNO3. 0.1% arginine .Dissolve
in water. (Professor A)

VII. GENERALIZATION

The manner in which proteins change their structure within the sight of specific synthetic
concoctions, acids or bases - protein denaturation - assumes a key job in numerous significant
natural procedures. What's more, the manner in which proteins communicate with different basic
particles is basic to discovering new medications. Microfluidic frameworks are touted as promising
frameworks for high-throughput examination of exceptionally little example volumes, with
applications in medication disclosure, therapeutic diagnostics, sedate detailing, biosensing and
manufactured science.

VIII. REFERENCES

Greenleafable, (n.d) Retrieved from https://brainly.com/question/2279077

Namrata Chhabra, Practical Biochemistry – Viva- Protein precipitation occurs. (dec. 1, 2011).
Retrieved from http://www.namrata.co/practical-biochemistry-viva-protein-precipitation-
reactions/

P.David Eckersall, Chapter 5 - Proteins, Proteomics, and the Dysproteinemias (n.d). Retrived
from https://www.sciencedirect.com/science/article/pii/B9780123704917000052

Professor A, What is the colored precipitate obtained in the sulfur test (lead acetate test)? (n.d)
Retrieved from
https://www.askmefast.com/What_is_the_colored_precipitate_obtained_in_the_sulfur_test_lead
_acetate_test-qna8994117.html#q3752828

Why tannic acids are used in the treatment of burns? (n.d) Retrieved from
https://www.answers.com/Q/Why_tannic_acids_are_used_in_the_treatment_of_burns

IX. DOCUMENTATION

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 Results from different test
Result of albumin in blurred test

Protein solutions reactions from

alcohol

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