BIOCHEMISTRY (MC2) LABORATORY ACTIVITY

no. 6
AMINO ACIDS AND PROTEINS

Submitted by: GROUP 4 (Vince Agustin, Cyenna

Norein Sagucio, Rica May Andres, Erich Bugarin)
Submitted to: Ms. Grace Seggay
 The isolation of proteins is a fundamental technique in biochemistry and food science,
allowing us to study the properties and functions of various proteins found in different
sources. This report outlines the procedures for isolating three distinct types of proteins:
gluten from wheat flour, bean protein (mongo), and egg albumin from eggs. Each method
highlights the importance of specific techniques such as washing, precipitation, and
evaporation, which is essential for obtaining pure protein extracts.
The first method involves the isolation of gluten, a vital protein complex in wheat
flour, through a process that including kneading and washing to remove starch. The resulting
crude gluten serves as a mixture of proteins that can be further analyzed. The second method
focuses on extracting protein from mongo beans, where soaking and grinding facilitate the
separation of soluble proteins from insoluble materials. The addition of acetic acid
precipitates the proteins, allowing for their collection and drying. Lastly, the preparation of
egg albumin emphasizes the importance of filtration and evaluation in obtaining a
concentrated protein product from egg whites.

BEAN PROTEIN (MONGO) (Biuret & Millons Test)

(Xantoproteic & Hopkins-Cole Test)

CASEIN (WHEAT FLOUR) EGG ALBUMIN (EGG)

A. Biuret Test
 The Biuret Test is a fundamental experiment in biochemistry that allows students to
detect the presence of proteins in various substances. focusing on the interactions of
the Biuret reagent with different protein sources. The Biuret reagent, composed of
sodium hydroxide (NaOH) and copper sulfate (CuSO4), is known for its colorimetric
response to proteins. In this experiment, we prepared a protein suspension using
powdered egg albumin, bean protein, and casein. The first step involved adding 1 ml
of 2.5 NaOH to 3 ml of the protein suspension and mixing thoroughly. This alkaline
environment is essential for the subsequent reaction with copper ions. After mixing, a
drop of 0.01M CuSO4 was added. The resulting color changes were meticulously
observed.
B. Xantoproteic Test
 The Xanthoproteic Test is a chemical test used to detect the presence of proteins in a
sample.
Test Procedure:
1. Add 1-2 drops of concentrated nitric acid to the sample.
2. Observe for a yellow color (xanthoproteic acid) indicating protein presence.
3. Heat the sample gently to intensify the color.

The test relies on the reaction between nitric acid and proteins, producing
xanthoproteic acid, a yellow-colored compound.
C. Millons Test
 Millon’s test is predicated on the principle of nitrification of the phenol group in
tyrosine, which then forms complexes with significant metals like mercury. A reagent
may be a compound or mixture added to a system to begin or check a chemical
change. A reagent may be used to confirm the presence or absence of a selected
chemical substance as the binding of reagents to the substance or different connected
substances triggers bound reactions. The reagent used for the test is Millon’s reagent,
consisting of metal nitrate of mercury and mercuric nitrate that is dissolved in
concentrated nitric acid.
D. Hopkins – Cole Test
 The Hopkins-Cole test, also known as the glyoxylic acid reaction, is a qualitative
assay used to detect the presence of tryptophan in proteins. This test, developed by
Frederick Gowland Hopkins and Sydney W. Cole in 1901, relies on the reaction
between the indole group of tryptophan and glyoxylic acid in the presence of
concentrated sulfuric acid, resulting in a characteristic purple color at the interface of
two liquid layers. The test is significant for its ability to confirm the presence of
tryptophan-containing proteins.
I. EGG ALBUMIN FROM EGG
A. Biuret Test
- The first protein tested was egg albumin. Initially, the solution appeared clear, but
upon adding the CuSO4 (cupric acid), a remarkable transformation occurred. The
solution first turned blue, indicative of the presence of copper ions, and subsequently
transitioned to violet. This violet color confirmed a positive result for proteins,
specifically indicating the presence of peptide bonds in the egg albumin, which
interact with the copper ions.
B. Xantoproteic Test
- The Xantoproteic test is a chemical test used to detect the presence of proteins in a
sample. In this experiment, 3ml of egg albumin was tested using concentrated nitric
acid (HNO3) as the reagent. The procedure involved adding 1 ml of concentrated
HNO3 to 3ml of egg albumin. Initially, the color of the mixture was clear. However,
upon heating, the mixture turned slightly yellow, indicating a positive result for
protein presence.
- The reagent, concentrated HNO3, reacts with proteins in the sample to produce a
yellow-colored compound, characteristic of proteins containing aromatic rings, such
as tyrosine and tryptophan. This reaction is evident in the color change observed,
where the formation of xanthoproteic acid results from the nitration of protein
molecules.
- In conclusion, the Xantoproteic test successfully demonstrated the presence of
protein, specifically albumin, in the egg albumin sample. The reaction between HNO3
and protein molecules resulted in a characteristic yellow coloration, indicating a
positive result.
C. Millons Test
- In egg albumin, we dropped more than 5 drops of reageant to the 3 ml of the protein
suspension. It was a clear color when it started then turn into foggy. Turn to pink to
dark red and it was insoluble and has a positive test.
D. Hopkins – Cole Test
- In the case of egg albumin, mix of 2 ml Hopkins-Cole reagent to 3 ml of the protein
suspension. Then add slowly 5 ml of concentrated H2SO4 (sulfuric acid). The initial
observation showed three distinct layers: a dark brown outer layer, a white central
layer, and floating particles. The test resulted in a negative outcome, indicating that
tryptophan was not present.
II. BEAN PROTEIN (MONGO)
A. Biuret Test
- We examined bean protein. The initial color of the solution was a striking neon green.
Following heating, the mixture developed distinct layers: a brown sediment at the
bottom, a greener middle layer, and a blue top layer. This complex layering suggested
the presence of various protein components, but the overall result was ambiguous.
The vibrant colors indicated the complexities of the bean protein composition, yet no
clear positive result was achieved.
B. Xantoproteic
- We conducted the Xantoproteic test on a 3ml bean protein sample, using 1ml of nitric
acid (HNO3) as the reagent. The initial color of the sample was yellow. Upon heating,
the color changed to a slightly orange hue, as can be shown by the picture where a
distinct coloration occurred.
- The reagent, nitric acid, reacts with proteins containing aromatic rings (tyrosine and
tryptophan) to produce a yellow-orange compound, xanthoproteic acid. This color
change indicates the presence of these amino acids in the bean protein sample.
- However, our observation differs slightly from the expected result stated in the
laboratory manual, which indicates an intensely yellow color. Instead, we observed an
orange color. This discrepancy may be due to:
- Variations in sample preparation or reagent concentration
- Differences in heating conditions or duration
- Presence of other substances influencing the reaction
- Despite this difference, the orange color still signifies:
- Presence of tyrosine and tryptophan amino acids in the bean protein
- Relatively high concentration of these amino acids
- Even distribution throughout the sample
- This positive result confirms protein presence in the bean protein sample. Further tests
are necessary to determine the exact type of protein.
C. Millons Test
- In bean protein , we also dropped more than 5 drops of regeant to the 3 ml of the
protein suspension. Insoluble but turn into dark pink if there's a lot of regeant. So it
was a positive. It was started with the white color and the drop of millons regeant it
turn into crystal inside then when it start boiling it , they turn into insoluble and pink
color.
D. Hopkins – Cole Test
- The bean protein we also mix 2 ml of Hopkins-Cole reagent to 3ml of the protein
suspension, and add slowly 5ml sulfuric acid, letting it flow down. The sample
exhibited three layers as well, with foam at the top, an orange second layer, and a
 black central portion. Similar to egg albumin, this test also yielded a negative result
for tryptophan.
III.

CASEIN FROM WHEAT FLOUR

A. Biuret Test
- Finally, we tested casein. The solution began as a brown color and then shifted to a
blue-green hue upon the addition of CuSO4. This color change was less vibrant than
that of the egg albumin and ultimately suggested a negative result for protein
presence. The blue-green coloration indicated that while some interaction with the
reagent occurred, it did not confirm the presence of proteins in the same manner as the
egg albumin.
B. Xantoproteic Test
- We performed the Xantoproteic test on a 3ml Casein sample, adding 1ml of nitric acid
(HNO3). Initially, the sample was black and insoluble, characteristic of Casein's
hydrophobic properties. Upon heating, the sample underwent a distinct color change
to orange, indicating the presence of tyrosine and tryptophan amino acids. This
coloration is due to the nitric acid reacting with aromatic rings in these amino acids,
producing xanthoproteic acid. The orange hue signifies a relatively high concentration
of tyrosine and tryptophan, evenly distributed throughout the sample. Although the
initial black color may be attributed to impurities or additives, the positive result
confirms protein presence in the Casein sample, as can be shown by the picture where
a distinct coloration occurred. Further tests are necessary to determine the exact type
of protein and characterize its properties.
C. Millons Test
- On casein, we also dropped again 5 drops of regeant. Before the color is black then
become yellowish. It has a particle and it was negative.
D. Hopkins – Cole Test
 - For casein extracted from wheat flour, we mix 2 ml of Hopkins-Cole reagent to 3ml
of the protein suspension, and add slowly 5ml sulfuric acid, letting it flow down. The
initial color was black, transitioning to light brown. However, this sample also
produced a negative result for tryptophan presence.

SOLUBILITY
This solubility has approximately 0.1 g of powdeed egg albumin, bean protein and
caesin(flour protein) in 1 ml of the solvents like water, saturated NaCl , 0.1 N HCl, and 0.1
NaOH. And then first we place about 0.1 of the protein in a dry test tube and heat in a
boiling water for 5 minutes. We cool and and we test its solubility with each solvent. We
repeat the process but we put 1 ml of water to the egg albumin before heating inthe water
bath.
A. Distilled Water
B. Saturated NaCl
C. HCl
D. NaOH

I. EGG ALBUMIN
- In egg albumim non heating process, the rusult of this with then solvent of distilled
water, Saturated NaCl, O.1N HCl, and 0.1 N NaOH are insoluble. While in dry
heating process, the result with solvent distilled water and saturated NaCl are
Insoluble and meanwhile the solvent 0.1 HCl and NaOH are soluble.On heating with
 distilled water process, the solvent distilled water, Saturated NaCl, and 0.1N NaOH
are insoluble, while on solvent 0.1N HCl is soluble.
Non – heating Dry heating
II. BEAN PROTEIN
- In mongo bean,the solvent Distilled water, Saturated NaCl and 0.1N NaOH are
Insoluble while on solvent. 0.1N HCl is soluble.

III. CASEIN
- On caesin wheat flour, the all solvent like, Distilled water, Saturated NaCl, 0.1N HCl
and 0.1N NaOH are insoluble