Experiment no.

Proteins

I. Objective(s)

The objectives of this experiment is to be able to know whether proteins can be denatured
with the addition heat, alcohol, strong acid, heavy metal salts, alkaloidal reagents, etc. Also,
to be able to know how proteins can be determined through various color tests.

II. Definition of Terms

COAGULATION – a process which involves coming together of colloidal particles to change

into large sized particles, which ultimately settle as a precipitate or float on the surface.

DENATURATION - a process in which proteins or nucleic acids lose the quarternary, tertiary,
and secondary structures which is present in their native state, by application of some
external stress or compound.

DERIVATIVES - a compound that is derived from a similar compound by

a chemical reaction

PRECIPITATE - the creation of a solid from a solution

REAGENT - a substance or compound added to a system to cause a chemical

reaction, or added to test if a reaction occurs

III. Theory Behind the Experiment

Proteins are large biomolecules, or macromolecules, consisting of one or more

long chains of amino acid residues. The protein sample used in this experiment
was the egg albumin, also known as, Ovalbumin (abbreviated OVA[1]), which is
the main protein found in egg white, making up approximately 55% of the total
protein.[2] The function of ovalbumin is unknown, although it is presumed to be
a storage protein.

The first three parts of the experiment explores the various ways of denaturing
proteins. Denaturation of proteins involves the disruption and possible
destruction of both the secondary and tertiary structures.

In tertiary structure there are four types of bonding interactions between "side
chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar
hydrophobic interactions. which may be disrupted. Therefore, a variety of
reagents and conditions can cause denaturation. The most common observation
in the denaturation process is the precipitation or coagulation of the protein.
Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic
interactions. This occurs because heat increases the kinetic energy and causes
the molecules to vibrate so rapidly and violently that the bonds are disrupted. The
proteins in eggs denature and coagulate during cooking. Other foods are cooked
to denature the proteins to make it easier for enzymes to digest them.

Moreover , Hydrogen bonding occurs between amide groups in the secondary

protein structure. Hydrogen bonding between "side chains" occurs in tertiary
protein structure in a variety of amino acid combinations. All of these are
disrupted by the addition of another alcohol. Alcohol denatures proteins by
disrupting the side chain intramolecular hydrogen bonding. New hydrogen bonds
are formed instead between the new alcohol molecule and the protein side
chains.

Proteins are known to possess salt bridges. Salt bridges result from the
neutralization of an acid and amine on side chains. The final interaction is ionic
between the positive ammonium group and the negative acid group. Any
combination of the various acidic or amine amino acid side chains will have this
effect.

As might be expected, acids and bases disrupt salt bridges held together by ionic
charges. A type of double replacement reaction occurs where the positive and
negative ions in the salt change partners with the positive and negative ions in
the new acid or base added. This reaction occurs in the digestive system, when
the acidic gastric juices cause the curdling (coagulating) of milk.

Correspondingly, Heavy metal salts act to denature proteins in much the same
manner as acids and bases. Heavy metal salts usually contain Hg+2, Pb+2,
Ag+1 Tl+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. Heavy metals may also
disrupt disulfide bonds because of their
high affinity and attraction for sulfur and will also lead to the
denaturation of proteins. Wherein, Disulfide bonds are formed by oxidation of
the sulfhydryl groups on cysteine.

For the last part of the experiment, various color tests were used to determine the
presence of amino acids and the different properties of the protein in egg albumin.
The principle of each color test are as follows:
The Ninhydrin test is due to a reaction between an amino group of free amino acid and ninhydrin.
Ninhydrin is a powerful oxidizing agent and its presence, amino acid undergo oxidative deamination
liberating ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin ( hydrindantin).
The NH3 formed from an amino group reacts with another molecule of ninhydrin and is reduced
product (hydrindatin) to give a blue substance diketohydrin ( Ruhemann’s complex). However, in
case of amino acid like proline and hydroxyproline, a different product having a bright yellow color is
formed. Asparagine, which has a free amide group, reacts to give a brown colored product.

 Biuret test is a general test for compounds having a peptide bond. Biuret is a compound
formed by heating urea to 180C. When biuret is treated with dilute copper sulfate in alkaline
condition, a purple colored compound is formed. This is the basis of biuret test widely used for
identification of proteins and amino acids.

 Xanthoproteic test is used to detect amino acids containing an aromatic nucleus (tyrosine,
tryptophan and phenylalanine) in a protein solution which gives yellow color nitro derivatives
on heating with conc. HNO3. The aromatic benzene ring undergoes nitration to give yellow
colored product. Phenylalanine gives negative or weakly positive reaction though this amino
acid contains aromatic nucleus because it is difficult to nitrate under normal condition. On
adding alkali to these nitro derivative salts, the color change from yellow to orange.

For Millon’s test, Compounds containing hydroxybenzene radical react with Millon’s reagent to form
red complexes. The only amino acid having hydroxybenzene ring is tyrosine. Thus, this test is
specific for the amino acid tyrosine and the protein containing this amino acid. Tyrosine when
reacted with acidified mercuric sulphate solution gives yellow precipitate of mercury-amino acid
complex. On addition of sodium nitrate solution and heating, the yellow complex of mercury-amino
acid complex converts to mercury phenolate which is in red color.

For the Hopkin’s-Cole test, The indole group of tryptophan reacts with glyoxylic acid in the presence
of conc. H2SO4 to give a purple colored complex. Glyoxylic acid is prepared by reducing Oxalic
acid with magnesium powder or sodium amalgam. Glacial acetic acid which has been exposed to
the sunlight also contains glyoxylic acid and can thus be used for this test.

For the Sulfur test, When cysteine is heated with strong alkali like NaOH, some of the sulfur is
converted to sodium sulfide (Na2S) which can be detected by precipitation as lead sulfide (PbS)
from alkaline solution.

IV. Discussion of Results

In the first part of the experiment, various processes were involved in the coagulation of
proteins. The following results are as follows: 1mL of egg albumin solution was boiled gently, which
changed the solution’s color into an opaque white. Next,1 ml of egg albumin solution was diluted in
1 ml of distilled water, then conc. HNO3 was slowly poured on top of the albumin solution. This
formed a yellow cloudy precipitate between the 2 liquids. Subsequently, 1 ml of egg albumin mixed
with 2 ml denatured alcohol formed a white foamy precipitate. Lastly, 1ml of egg albumin with
drops of 0.10M NaOH only led to the formation of heat with no change in color and absence of
precipitate, whereas, 1ml of egg albumin with drops of saturated NaCl also resulted to no change in
color and absence of precipitate. Thus, it can be said that heat, HNO3 and denatured alcohol were
able to coagulate the proteins in the sample.
 For the second part of the experiment, proteins were precipitated with heavy metal salts. The
results are as follows: Egg albumin solution mixed with Pb(OAc)2 led to the formation of a white foamy
precipitate. Next, with the addition of HgCl2 to the egg albumin solution, a thin white foamy precipitate
formed. Lastly, egg albumin solution was also mixed with AgNO3, wherein a white-purple cloudy
precipitate formed, and the solution changed into a purple color. All the solutions added were able to
form precipitate in the egg albumin solution. Hence, heavy metal salts denature proteins.

Furthermore, in this experiment, proteins were also precipitated with alkaloidal reagents. All these
reagents were mixed with egg albumin solution. The results are as follows: The reaction with Tannic acid
produced a white compact or buff precipitate. Whereas, the reaction with Picric acid produced a white
cloudy precipitate and the reaction with Trichloroacetic acid produced a thin white cloudy precipitate.
All the solutions added were able to form precipitate in the egg albumin solution. Therefore, alkaloidal
reagents are also capable of denaturing proteins.

For the last part of the experiment, different color tests for proteins were conducted. The results are
as follows:

 For the Ninhydrin test, egg albumin was mixed with Ninhydrin reagent and was
heated to boiling. This produced a blue-violet color change, which indicates a
positive test. This color complex is said to indicate the presence of the -NH2
group.
 For the Biuret test, egg albumin was mixed with 10% NaOH and drops of 0.5%
CuSO4. It was observed that the solution changed into a violet color. This
indicates a positive result. Thus, it can be said that the egg albumin solution
contains 2 or more peptide bonds.
 For the Xanthoproteic test, egg albumin was mixed with concentrated nitric acid
and dropped with NaOH. This formed a yellow-orange colored precipitate. This
indicates a positive test due to the nitration of the benzene ring.
 For the Millon’s test, egg albumin was added with drops of Millon’s reagent,
which produced a white-yellow precipitate and coloration. This indicates the
presence of Tyrosine.
 For the Hopkin’s-Cole test, egg albumin was added with Hopkin’s-Cole reagent
and concentrated H2SO4, which produced a white foamy precipitate. This
indicates a negative result. The solution must form a violet colored complex in
order to determine the presence of the amino acid, tryptophan.
 For the sulfur test, egg albumin was mixed with 10% NaOH and dropped with
5% lead acetate. A black precipitate formed on the surface of the solution. This
indicates the presence of amino acid containing sulfhydral group (-SH), cysteine.
V. Conclusion
 Proteins are large biomolecules, or macromolecules,
consisting of one or more long chains of amino acid residues.

Proteins undergo denaturation, which involves the disruption and possible

destruction of both the secondary and tertiary structures. The most common
observations in the denaturation process is the coagulation and precipitation. the
protein will coagulate with the addition of heat, strong acid and alcohol. Whereas,
the protein will precipitate with the addition of heavy metal salts and alkaloidal
reagents. Moreover, no one test is specific for proteins. Thus, several color tests
are necessary in order to identify whether a substance is a protein.

VI. References
Karki, G. (2018, December 18). Ninhydrin Test: Principle, Requirements, Procedure

and Result. Retrieved from https://www.onlinebiologynotes.com/ninhydrin-test-

principle-requirements-procedure-and-result/

Karki, G. (2018, December 18). Xanthoproteic test: Objective, Principle, Reagents,

Procedure and Result. Retrieved from

https://www.onlinebiologynotes.com/xanthoproteic-test-objective-principle-reagents-

procedure-and-result/

Karki, G. (2018, December 18). Biuret test: Principle, requirements, Procedure and Result.

Retrieved from https://www.onlinebiologynotes.com/biuret-test-principle-requirements-

procedure-and-result/
 Karki, G. (2018, December 18). Lead sulfide test: Detection of amino acid containing

sulfhydral group (-SH). Retrieved from https://www.onlinebiologynotes.com/lead-

sulfide-test-detection-of-amino-acid-containing-sulfhydral-group-sh/

Karki, G. (2018, December 18). Millon's test: Objective, Principle, Reagents,

Procedure and Result. Retrieved from https://www.onlinebiologynotes.com/millons-

test-objective-principle-reagents-procedure-and-result/

Ophardt, C. E. (2003). Denaturation of Proteins. Retrieved from

http://chemistry.elmhurst.edu/vchembook/568denaturation.html

Ophardt, C. E. (2003). Denaturation of Proteins. Retrieved from

http://chemistry.elmhurst.edu/vchembook/568denaturation.html

Vid script

The objectives of this experiment is to be able to know the various ways on how proteins
can be denatured. Also, to be able to know how proteins can be determined through various
color tests.

Proteins are large Moleculd, consisting of one or more long chains

of amino acid residues. The protein used in this experiment was found
in the egg albumin solution, also known as,
Ovalbumin (abbreviated OVA[1]), which is the main protein found
in egg white, making up approximately 55% of the total protein.[2]

Proteins undergo denaturation. Denaturation of proteins involves the disruption and

possible destruction of both the secondary and tertiary structures. The most
common observation in the denaturation process is the precipitation or
coagulation of the protein.
Egg albumin coagulates with the addition of heat, strong acid, and alcohol. It also
forms precipitate when heavy metal salts and alkaloidal reagents are added to it.

Several color tests were done to determine amino acids and proteins. These
tests help identify certain structures present, which is necessary to know whether
a substance is a protein. All the tests in this experiment yielded positive in our
results. The following principles of the color tests are as follows:

the Ninhydrin test gives a blue-violet color complex, which indicates the presence of amino
acids

the biuret test give a violet complex indicates the presence of peptide bonds

the xanthoproteic test forms an orange ppt indicates the presence of proteins with aromatic acids

the millon’s test forms a yellowish ppt which indicates the presence of tyrosine

hopkin’s-cole test indicates the presence of tryptophan with the formation of a violet colored
complex

sulfur test forms a black ppt which indicates the presence of sulfur containing amino acids