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Biochemistry Lab Con Proteins

The document describes several experiments to test proteins using egg whites. It discusses how heat, alcohols, heavy metals and other compounds denature or precipitate proteins. Color tests are also described to detect proteins including Xanthoproteic, Biuret, Hopkins-Cole and Ninhydrin tests.

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riana santos
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220 views47 pages

Biochemistry Lab Con Proteins

The document describes several experiments to test proteins using egg whites. It discusses how heat, alcohols, heavy metals and other compounds denature or precipitate proteins. Color tests are also described to detect proteins including Xanthoproteic, Biuret, Hopkins-Cole and Ninhydrin tests.

Uploaded by

riana santos
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Experiment 1:

Proteins
By Allan Troi Ramos, Rianna Julianne Santos, Jasmine Vonne Saucelo, Ma. Cheeny Sy
Protein
Denaturation
process that alters the shape of a protein by disrupting the
secondary, tertiary, or quaternary structure.
High temperature, acid, base and agitation/stress can
denature a protein.
Compact water-soluble proteins uncoil and become less
water soluble
EFFECTS OF HEAT AND ALCOHOL
a. Place 2.0 mL of egg white solution
in a test tube and heat in a boiling
water bath for 5 minutes.
Compare the appearance of the
heated sample with the standard.
b. Label two tubes as 1 and 2. Add 2.0
mL of egg white solution to each

Procedure tube. To tube no. 1, add 95%


ethanol and to tube no. 2, 70%
ethanol. Compare the appearance
of the resulting mixtures with the
standard.
c. Record your observation as (-)
for no precipitation, (+) slight
precipitation (cloudiness), or (++)
for heavy precipitation.
PRINCIPLES

•modifying the conformation of the protein structures without


rupturing the native peptide linkages

•This inactivates the functionality of the protein molecules,


decreases its solubility, decreases/destroys its biological
activity, improves digestibility and alters the water binding ability
of the molecule.

•Denaturation of proteins is achieved by disrupting the


hydrogen bonding in the peptide linkage by applying external
stress
.
Alchohol Disrupts Hydrogen Bonding:
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.
•A 70% alcohol solution is used as a disinfectant on the skin. This
concentration of alcohol is able to penetrate the bacterial cell wall
and denature the proteins and enzymes inside of the cell.
•A 95% alcohol solution merely coagulates the protein on the
outside of the cell wall and prevents any alcohol from entering the
cell.
Results
1.Effects of Heat and Alcohol Test
for Turbidity

Observation
Heat Cloudy solution

95% ethanol More precipitate

70% ethanol Little precipitate


Effect of Heavy Metals
a. Add 2.0 mL of egg white
solution to each of the two
test tubes labeled 1 and 2.
b. To test tube !, add 1.o mL of
% of AgNO3 solution to

Procedure
each of the two test tubes
labeled 1 and 2.
c. Decant the supernatant
liquid and test the
solubility of a small
portion of the precipitate
in 5.0 mL of water.
Principle
Heavy Metal Salts:

•Heavy metal salts act to denature proteins in much the


same manner as acids and bases.

•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.
Results
Color of Solubility
Precipitate in Water

Silver White Insoluble


Nitrate

Lead White Insoluble


Acetate
Heavy Metal Ions Test: Heavy metal ions precipitate proteins from their solutions by cross-linking
free amino groups and carboxylate groups.

Ions commonly used for testing for the presence of proteins include Zn2+, Fe3+, Cu2+, Sb3+, Ag1+,
Cd2+, and Pb2+.

Among the metal ions, Hg2+, Cd2+, and Pb2+ have very high toxicity. They cause serious damage to
proteins (especially enzymes) by denaturing them. Victims who have swallowed Hg2+ or Pb2+ ions
are often treated with an antidote of a food rich in protein. The protein can combine with the
mercury and lead ions and minimize absorption of these ions. Milk and raw egg white are used
most often. The precipitated protein complexes are then immediately removed from the stomach by
an emetic.
Color Reactions
of Proteins and
Amino Acids
Xanthoproteic Test
a. Add 0.5 mL of concentrated
HNO3 to 1.0 mL of egg white
solution in a test tube.
b. Mix with a stirring rod and
warm in a water bath for 5

Procedure minutes. Note the color the


precipitate formed.
c. Cool the contents of the tube
and make it alkaline by adding
50% NaOH. Note the changes in
the mixture.
Principle
Xanthoproteic Test is used to detect Amino Acids containing in an Aromatic
nucleus Tyrosine and Tryptophan protein solution which gives yellow color
nitro derivatives on heating with conc. HNO3.
Principle
Although there is a presence of Aromatic nucleus in the Phenylalanine; it is
difficult to nitrate under normal condition hence, Phenylalanine gives a negative
or weakly positive reaction.
Results

The result of Concentrated


HNO3 with the Egg White
Solution.

Formed Yellow Precipitate in


color due to the nitration of
amino acids present.
The result of adding the
NaOH to the solution.

Formed a darker
yellow-orange precipitate
indicating the the test is
positive
Biuret Test
a. Mix 1.0 mL of egg white
solution and 10 gtts of 6M
NaOH in a test tube.
b. Add 1 drop of 0.5% CuSO4
solution. Mix well and record
your observation.

Procedure c. Dissolve one tablet of


aspartame or ½ sachet of Equal
in 2.0 mL of water.
d. Add the resulting solution 10
gtts of 6M NaOH and 0.5%
CuSO4 solution.
e. Mix well and record your
observation.
Principle
Biuret Test is based on the ability of Copper Ions to form a violet colored Chelate
Complex with peptide bonds in alkaline solution. The 4 Nitrogen atoms in the
peptide bonds coordinate a Cu Ion to form this complex.
Results
ASPARTAME SOL.

No reaction at all. The color


remained the same as it is.

Resulting the the test with


Aspartame is Negative.

EGG WHITE SOL..

The solution became deep


purple or violet in color.

Resulting that the test is


Positive.
Ferric Chloride Test
a. add 10 gtts of ninhydrin
solution to each of the two test
tubes, one containing the 2.0
mL of egg white solution and
the other with 2.0 mL of the
Procedure aspartame or Equal® aqueous
solution. Mix thoroughly.
b. Heat the tubes in a boiling
water bath until a color change
is observed.
c. Compare obtained results.
Principle
Ferric Chloride Test

● Color test for the presence of phenols (compound with a hydroxyl group linked directly
to a benzene ring) in a solution
● Phenols form a violet complex with Fe(III)+ (ferric oxide)
Results
Results were negative and flawed for
there was no change of color from it’s
original cream color
Hopkins-Cole Test
a. add 10 gtts of ninhydrin
solution to each of the two test
tubes, one containing the 2.0
mL of egg white solution and
the other with 2.0 mL of the
Procedure aspartame or Equal® aqueous
solution. Mix thoroughly.
b. Heat the tubes in a boiling
water bath until a color change
is observed.
c. Compare obtained results.
Principle
Hopkins Cole Test

● Color test for the presence of tryptophan in a sample


● Hopkins Cole reagent: magnesium glyoxolate
● Tryptophan: alpha-amino group, alpha-carboxylic acid group and side chain
indole)
● Tryptophan is present if an indole ring forms in a red and violet color
Results
Results were positive as there was a
visible separation of a violet indole ring
between the two solutions
Ninhydrin Test
a. Add 10 gtts of ninhydrin
solution to each of the two test
tubes, one containing the 2.0
mL of egg white solution and
the other with 2.0 mL of the
Procedure aspartame or Equal® aqueous
solution. Mix thoroughly.
b. Heat the tubes in a boiling
water bath until a color change
is observed.
c. Compare obtained results.
Principle
Ninhydrin test is generally utilized
to detect the presence of α-amino
groups (free amino acid). The
reaction between the ninhydrin and
amino acid produces Ruhemann’s
Purple.
Mechanism:

● Oxidative Deamination and


Decarboxylation
● Reduction of Ninhydrin
Principle
The ninhydrin test can be utilized to 19 of the 20 amino acid, with the exception
of proline and hydroxyproline (cyclic imino acid). Their α-amino group is part
of a five-member ring and do not react with the ninhydrin in the same way as
other amino acids producing a yellow color.
Results
● The aspartame aqueous solution
turned clear, while the egg white
turned into a deep blue solution.

● The bluish color signifies the


presence of free α-amino acid in the
egg white.
Sakaguchi Test
a. Strictly observed the order of
addition of the reagents. To the 5.0
mL of egg white solution, add 1.0
mL of 10% NaOH solution.
b. Mix, then add 1.0 mL of 0.2%

Procedure
α-naphthol solution. Mix
thoroughly.
c. After 3 minutes, add 5 drops of
sodium hypochlorite (bleach).
d. Immediately note the color of the
resulting solution as it fades
quickly. Record your observation.
Principle
Sakaguchi test is a specific test for the Guanidino group. The guanidine group
reacts with the Sakaguchi reagent (α-naphthol and sodium hypochlorite) to
form a red-colored complex.
Results
● The egg white solution turned red,
which fades quite easily, signifying
the presence of a Guanidino group.
Lead Acetate Test
a. Add 1.0 mL of 50% NaOH
solution to a test tube containing
2.0 mL of egg white solution,
then mix.

Procedure
b. Add 5 drops of lead acetate
solution.
c. Mix and heat the test tube in a
boiling water bath until a change
in color is observed. Record your
observation.
Principle
Lead Acetate Test is used to detect the thiol group in cysteine (monomer) or
cystine (dimer). When cysteine is heated with strong alkali (NaOH), some
sulphur is converted to sodium sulphide (Na2S), which can be detected by
precipitating it to lead sulphide (PbS) with the addition of lead acetate
(Pb(CH3COO2)) solution.

sulfur in cysteine + 50% NaOH → heating→ Na2S

Na2S + Pb(CH3COO)2 → PbS(black ppt) +Na2(CH3COO)2


Principle
NaOH acts as a hydrolyzing agent that frees the sulphide group of the amino
acid forming sodium sulfide, which could then be detected by the presence of
brown/black precipitate with lead acetate.

Methionine does not give a positive lead acetate test because it sulfur does not
split in the presence of alkali.
Results
● Black precipitate indicates the
presence of sulfhydril (-SH) group.
End of Discussion.
Thank you for Listening!

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