CHEM 132.

2 – BIOCHEMISTRY (LABORATORY)
LABORATORY REPORT

Name _John Allan T. Pasana_______ Date Submitted: _01/26/21___

Lab Schedule: MTh at 10:00-12:00_____ Date Performed: _01/26/21___

Exercise No. 6
Proteins
I. OBJECTIVES:
 To identify the structural patterns of proteins.
 To use the isoelectric point of casein in milk to isolate the protein.
 To use chemical tests to identify amino acids and proteins.
 To observe the denaturation of proteins.

II. RESULTS AND DISCUSSION:

A. SEPARATION OF A PROTEIN (CASEIN) FROM MILK

Mass of flask (g) 82.1433g

Mass of flask and milk (g) 132.8872g
Mass of milk (g) 50.7439g
Mass of Casein product (g) 4.6983g
% casein in milk 9.259% or 9.26%

Discussion:

In this experiment, an Erlenmeyer flask was weighed and its mass was recorded (which is
82.1433g). Next, 50 mL non-fat milk was then added to the flask and reweighed (which resulted to
132.8872g). To get the mass of the milk itself, the mass of the flask was subtracted from the mass
of the flask and milk, and that is exactly why we got 50.7439 as our milk’s mass. Moving on the
flask was placed in a hot water bath made from a 400-mL beaker. The temperature of the milk was
then held at about 50℃. 10% acetic acid was also slowly added drop-wise. When the casein
reaches its isoelectronic point, it becomes insoluble, and precipitates out leaving a clear solution.
Furthermore, when there is no further precipitation that occurs, stop adding acid and let the
sample cool. We then weighed the filter paper which is 1.2321g before using it to filter the
precipitate. The filtration apparatus was then utilized to collect protein particles. The protein was
dried on the filter paper and was later on measured (which resulted to 5.9304g). To get the mass
of the casein product, I simply subtracted the mass of the filter paper from the mass of the dried
filter paper with the casein in it, and finally obtained 4.6983g as the casein product’s mass. Finally,
to get the % casein in milk, I basically divided the mass of casein (4.6983g) by the mass of milk
(50.7439g) and multiplied it by 100, and finally got 9.259% which I rounded to 9.26% and that is
the % casein in milk.

TEST FOR AMINO ACIDS AND PROTEINS

B. Biuret Test

Compound Color Conclusions

Glycine Blue Negative for Biuret Test

Alanine Blue Negative for Biuret Test

Glutamine Blue Negative for Biuret Test

Aspartic acid Blue Negative for Biuret Test

Deep Blue-Violet with Positive for Biuret Test
Albumin
formation of precipitate
Casein Blue Negative for Biuret Test

Discussion:
Biuret test is used for detecting compounds with peptide bonds. A biuret reagent may be used
to test the aqueous sample. This blue reagent is made by combining sodium hydroxide and copper
sulfate solutions. A few drops of this reagent will turn the aqueous sample containing compounds
with peptide bonds from pale to intense violet colour. The violet colour intensity depends on the
number of peptide bonds in the sample. This test is also used to detect proteins. It is because
proteins are made up of polypeptides, which in turn, are made of amino acids joined by peptide
bonds. The longer the peptide bonds there are, and therefore, the more intense the violet color will
be when biuret test is applied. After the experiment, it was found out that only Albumin tested
positive for this test. It is because the color of the solution is deep blue and approaching the shade
of violet. It can also be seen that there is a formation of white precipitate. This white precipitate itself
is an indication for the presence of Albumin while its filtrate depicts that it contains additional
proteins. Glycine, Alanine, Glutamine, and Aspartic Acid, on the other hand, tested negative for this
test since these 4 are amino acids and not proteins. Casein, on a similar note, even though it is a
protein, tested negatively for this test. This may be associated to the lack of its protein composition
or the presence of free amino acids (without peptide bonds).

C. Ninhydrin Test
 Compound Color Conclusions
Glycine Deep Blue to Purple Positive for Ninhydrin Test

Alanine Deep Blue to Purple Positive for Ninhydrin Test

Deep Blue to Purple on the
upper part while a speck
Tyrosine Positive for Ninhydrin Test
of purple is shown
underneath it
Glutamine Deep Blue to Purple Positive for Ninhydrin’s Test
Purple on the upper part Positive for Ninhydrin’s Test
Albumin while a white color is
observed underneath it
Aspartic acid Deep Blue to Purple Positive for Ninhydrin’s Test

Casein Deep Blue to Purple Positive for Ninhydrin’s Test

Discussion:
Ninhydrin test is a chemical test performed to detect the presence of ammonia,
primary/secondary amines, or amino acids. This test involves the addition of ninhydrin
reagent to the test sample that results in the formation of deep blue color, often termed as
Ruhemann’s purple, in the presence of an amino group. After this experiment, it was found out
that Glycine, Alanine, Tyrosine, Glutamine, Albumin, Aspartic acid and Casein all had positive
results for this test. It is because these 7 had a deep blue to purple color which indicates the
presence of free amino acids in them.

D. Xanthoproteic Test

Compound Color Conclusions

Valine Clear Negative for Xanthoproteic Test
Yellow approaching
Tyrosine Positive for Xanthoproteic Test
Orange
Glutamine Clear Negative for Xanthoproteic Test

Aspartic acid Clear Negative for Xanthoproteic Test

Albumin Clear Negative for Xanthoproteic Test

 Casein Clear Negative for Xanthoproteic Test

Discussion:
The Xanthroproteic test is specific for the R groups of tyrosine and tryptophan. Amino acids
or proteins containing aromatic rings will react with concentrated nitric acid to give nitro-
substituted benzene rings which appear as yellow-colored products. Since this test is specific
to tyrosine and tryptophan, it is very obvious that in among the different compounds, only
tyrosine would yield a positive result. Out of all the compounds, it is the only one which had a
yellow color which is near to orange. This basically means that tyrosine contain an aromatic
group. On the addition of alkali, however, the residue turns orange due to the formation of a
salt of the tautomeric form of the nitro compound. Benzene ring-containing amino acids like
phenylalanine don’t give a positive test to this test because the phenyl group in phenylalanine
is very stable, which doesn’t react with nitric acid in the conditions of this test. However,
phenylalanine might give a positive result after an extended period of heating. On the other
hand, the other compounds didn’t give a positive result since they don’t contain an aromatic
side chain.

E. Sulfur Test

Compound Color Conclusion

Clear with white
Alanine Negative for Sulfur Test
precipitate
Lysine Clear Negative for Sulfur Test
Clear with a speck of
Glutamine orange precipitate Negative for Sulfur Test
underneath
Light Brown on the upper
part, having the presence
Albumin of black precipitate in the Positive for Sulfur Test
middle and white
precipitate underneath it
Clear with a bit of black Positive for Sulfur Test
Casein
precipitate on the bottom

Discussion:
 This test is used to detect amino acids which contains sulfur group on their chain. Solutions
of protein containing cystine, cysteine and methionine, when heated with NaOH, splits up the
sulfur to form Na2S.. This when treated with lead acetate produces a black precipitate due to
the formation of lead sulphide (PbS). After the experiment was conducted, it was found out
that only albumin and casein tested positive for it since they both have a formation of black
precipitate in them. This indicates the presence of cysteine or cystine. On the other hand,
alanine, lysine, and glutamine tested negative for sulfur test due to no formation of black
precipitate. This depicts that there is an absence of cysteine or cystine in these compounds.

F. Denaturation of Proteins
Denaturing Agent Observations
Heat Coagulation clumps of white solid, curdling,
and formation of opaque white color
Heavy metal: AgNO3 Formation of a creamy white-colored liquid
Heavy metal: PbAc2 Formation of a creamy white-colored liquid
Strong acid (HNO3) Creamy white-chunks or clumps
Alcohol (Ethanol) No color change, formation of thick creamy
liquid on top

Discussion:
Denaturation of protein occurs when the tertiary or quaternary structure of the protein is
altered or destroyed. In many cases, coagulation of the protein occurs. Agents causing
denaturation include heat, acid, base, ethanol, tannic acid, and heavy metal ions of silver, lead,
and mercury. Heat increases the vibrations of the atoms which disrupt the hydrogen bonding
and hydrophobic (nonpolar-nonpolar) bonds. Strong acids and bases add or remove hydrogen
ions to the ionic side groups. As a result, salt bridges (ionic bonds) are broken. Alcohol is an
organic solvent that disrupts the hydrogen bonding of the secondary and tertiary protein
structures. The heavy metal ions, Ag+ , Pb2+, and Hg2+, react with the disulfide bonds of the
tertiary structures and the carboxylic groups of the acidic R groups. These are the reasons why
the denaturation off egg albumin in these agents produced these observations of mine.
III. CONCLUSIONS:

Proteins are the end products of the decoding process that starts with the information in
cellular DNA. As workhorses of the cell, proteins compose structural and motor elements in the
cell, and they serve as the catalysts for virtually every biochemical reaction that occurs in
living things. This incredible array of functions derives from a startlingly simple code that
specifies a hugely diverse set of structures. In fact, each gene in cellular DNA contains the code
for a unique protein structure. Not only are these proteins assembled with different amino acid
sequences, but they also are held together by different bonds and folded into a variety of three-
dimensional structures. The folded shape, or conformation, depends directly on the linear
amino acid sequence of the protein. The primary structure of a protein — its amino acid
sequence — drives the folding and intramolecular bonding of the linear amino acid chain,
which ultimately determines the protein's unique three-dimensional shape. Hydrogen bonding
between amino groups and carboxyl groups in neighboring regions of the protein chain
sometimes causes certain patterns of folding to occur. Known as alpha helices and beta sheets,
these stable folding patterns make up the secondary structure of a protein. Most proteins
contain multiple helices and sheets, in addition to other less common patterns. The ensemble
of formations and folds in a single linear chain of amino acids — sometimes called
a polypeptide — constitutes the tertiary structure of a protein. Finally, the quaternary
structure of a protein refers to those macromolecules with multiple polypeptide chains or
subunits. The different tests above which served different purposes are very important as it
gives essence and provides ways for us to apply the concept of proteins in different scenarios.
Moreover, the way proteins change their structure in the presence of certain chemicals, acids
or bases - protein denaturation - plays a key role in many important biological processes. And
the way proteins interact with various simple molecules is essential to finding new drugs to
cure different ailments and health conditions.