I.

Name:
(GROUP 5)
ORTONIO, Eunice Jireh F. (Leader)
LLANILLO, Piolo Angelo E.
MOPAL, Gerhard D.
NARVAZA, Donna Daniela A.
PABLICO, Jessica Lorenz M.
PAGUIO, Norhana T.
PIZARRAS, Hannah Gale G.

II. Title of experiment and no.: THE PROTEIN EXPERIMENT NO. 2A

III. Objectives:
• To perform qualitative test for different types of proteins
• To identify proteins based on the different tests performed
• Relate the test results to the chemical structure of each protein or amino acid

IV. Theoretical Background:

The Biuret test is a chemical assay that detects the presence of proteins in a sample. The test relies on a color change to
confirm the presence of proteins. If proteins are found, the sample will turn violet. he biuret test uses an alkaline mixture, or
reagent, composed of potassium hydroxide and copper sulfate. The normal color of biuret reagent is blue. The reagent turns violet
in the presence of peptide bonds -- the chemical bonds that hold amino acids together. The proteins detected must have at least
three amino acids, which means that the protein must have at least two peptide bonds. The reagent’s copper ions, with a charge of
+2, are reduced to a charge of +1 in the presence of peptide bonds, causing the color change (Bank. n.d.)

Ninhydrin test is a general test and thus given by all amino acids. This test is due to a reaction between a 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 a amino group reacts with another molecule of ninhydrin and is reduced product ( hydrindatin) to give a blue
substance diketohydrin ( Ruhemanns complex). However, in case of imino 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
(https://www.onlinebiologynotes.com, 2019)

Hopkins-Cole test is used to detect amino acid tryptophan present in protein. 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.

The Sakaguchi Test or the arginine contents of a variety of protein samples determined by the quantitative Sakaguchi method
were in good agreement with the values obtained by quantitative ion-exchange chromatography, after acidic hydrolysis. One
protein, α-chymotrypsin, while possessing a linear relationship between the colour obtained in the Sakaguchi test and the protein
concentration, gave colour yields which corresponded to the apparent presence of an additional Sakaguchi-positive residue. This
test is specific for free Arginine or Proteins containing arginine. Sakaguchi’s test is positive for the amino acid reacts with A-
Naphtol and Alkaline Hypobromite to give red-coloured complex.

The 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 fro yellow to orange.
(https://www.onlinebiologynotes.com)

V. Data and Results

 TEST ALBUMIN ASPARTAME EVAPORATED MILK

HOPKINS-
COLE TEST

BIURET
TEST

SAKAGUCH
I TEST
TEST ALBUMIN/ ASPARTAME/ EVAPORATED MILK

NINHYDRIN TEST

XANTHOPROTEIC TEST
VI. Data Analysis

This section consist of all the data and results of the experiment. Every test must used 3 proteins samples which is the egg
albumin, aspartame or the equal and lastly the evaporated milk. These tests helps us to identify the different types of amino acids
present in the protein samples. The Biuret test determined if there is peptide bonds present in the sample if the sample turns color
violet.the only samples where turned into violet are the evaporated milk and the albumin and it means that it is positive indicator
but the albumin shows darker color so it means it is strong and more peptide bonds in the sample while the aspartame shows like
a yellowish orange. The Ninhydrin test have no positive indicators like all of its samples, putting solution in the samples doesn't
make its color change. In the Hopkins-Cole test the evaporated milk only shows positive indicator because of the appearance or
the color of the sample because it turned Into violet and it has a ring between its layers while the albumin and the aspartame
shows negative indicator because it like whitish color and no rings on it. In the sakaguchi test, the aspartame shows a yellow
color and the albumin showed a dark color while in the evaporated milk only appeared in the top of the sample. In the
Xanthoproteic test, albumin and evaporated milk shows a positive indicator because the nitric acid was poured into the samples
but the aspartame when it was poured with the nitric acid it becomes colourless and its just like nothing but when dropping a
litmus paper in the samples all of them turns Ito blue.

VII. Conclusion

As for the conclusion, the qualitative test in different types of proteins was able to show the presence of proteins. The
proteins are are dissident which have been completed and have been made available of the group . Due to amino acids are the
monomers that make up proteins. Specifically, amino acid have been made up of test results and processes have been deduced of
process in all subject combined. The observation was recorded.

VIII. Reference

https://www.onlinebiologynotes.com/sakaguchi-test-objective-principle-reagents-procedure-and-result/

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

https://slinkyscribblets.weebly.com/biochem-lab/activity-no-5-color-tests-for-proteins-and-specific-amino-acids