BSN – 1C

GROUP 5

EXPERIMENT NO. 2A

Leader: Requerme, Ela Evan

Members:

Nesperos, Jethro

Pansoy, Kyla Nico

Parra, Crizza

Pascua, Kristine Ysabel

Ranes, Franz Louise

Saballa, Kenneth Joshua

I. INTRODUCTION

Proteins are highly complex substance that is present in all living organisms. Proteins are the
building blocks of our muscle mass, it is the basic component of living cells and made up of carbon,
hydrogen, oxygen, nitrogen and amino acids.

BACKGROUND

PURPOSE

IMPACT

II. OBJECTIVES

The objective of this study aims to attest which of the test can detect protein and which can
detect specific amino acids in a protein molecule; thus, relating it to its chemical structures. The
qualitative test will be conducted in order to determine the different types of proteins.
III. METHODOLOGY

Materials:

Beaker Test tube holder

Graduated Cylinder Test tube brush
Stirring rod Hot plate
Test tube racks Water bath
Test tubes

Reagents:

Distilled water Hopkins-Cole reagent

50% NaOH 0.2% α-naphthol solution
6M NaOH Concentrated H2SO4
0.5% CuSO4 solution Sodium hypochlorite
Ninhydrin solution Concentrated HNO3

Protein Samples:

1 small tetra pack evaporated milk

1 fresh chicken egg
Aspartame or Equal (5 tabs or 1 sachet)
A. SAMPLE PREPARATION
Add 5ml of egg Mix
1. EGG ALBUMIN SOLUTION Beaker 45ml water
white

Add 5 tabs or 1 Dissolve

2. ASPARTAME OR EQUAL Beaker 15.0ml water
sachet

Add 10ml of Mix

3. EVAPORATED MILK Beaker 40ml water
evaporated milk

B. HOPKINS-COLE TEST C. BIURET TEST D. NINHYDRIN TEST

Test Tube Test Tube Test Tube

Add Add Add

2.0ml Hopkins- 1.0ml protein 10 drops

Cole reagent sample Ninhydrin solution
Mix
Mix Mix
1.0ml protein
2.0ml protein 10 drops of 6M
sample
sample sodium hydroxide
solution
Tilt and Mix
pour
Boiling water
2 drops of 0.5%
bath
copper sulfate
1.0ml
solution
concentrated
sulfuric acid Tabulate
Compare results
Hold the Record your
test tube observation

Observe the ring

formed. Note
the result.
E. SAKAGUCHI TEST F. XANTHOPROTEIC TEST

Test Tube Test Tube

Add Add

5ml protein sample 0.5ml concentrated

Mix nitric acid
Mix
1.0ml of 6M sodium
hydroxide solution 1.0ml protein
Mix sample
Heat
1.0ml of 0.2% α-
naphthol solution 5 minutes
Wait

3 minutes Observe the

Add color
Add
5 drops of sodium
chlorite
50% sodium
hydroxide

Immediately record
your observation
Observe the
changes
IV. RESULTS & DISCUSSION

RESULTS

CONTROL TEST OBSERVATION

EGG ALBUMIN SOLUTION No color changes occurred.

Remained colorless.
Acted as constant.

ASPARTAME No color changes occurred.

Remained colorless.
Acted as constant.

EVAPORATED MILK No color changes occurred.

Remained white in color.
Acted as constant.
 TESTS OBSERVATIONS RESULT

Egg & Milk: Separated and Egg & Milk: Positive

A. HOPKINS – COLE TEST formed two layers and a ring was
observed at the center. Colors
were purple and whitish cloud. Aspartame: Negative
Aspartame: None.
Egg & Milk: The color changed Egg & Milk: Positive
into violet.
B. BIURET TEST
Aspartame: Colorless, Aspartame: Negative
transparent.
Egg & Milk: The color changed Egg & Milk: Positive
C. NINHYDRIN TEST into purple.

Aspartame: Crystal clear blue. Aspartame: Negative

Egg: Red color was observed, the Egg: Positive
D. SAKAGUCHI TEST color went to the bottom.
Milk: Red color stayed at the Milk: Positive
center.
Aspartame: Light green was Aspartame: Negative
observed.
Orange colored lower layer,
E. XANTHOPROTEIC TEST yellowish cloud top layer.
Positive
Aspartame: Clear with a less
pigmented yellow liquid.
DISCUSSION

During the Hopkins- Cole Test, egg and milk did changed in color after the concentrated sulfuric
was added, a purple ring appeared between the two layers. This test is used for detecting the presence
tryptophan in proteins. According to Fearon (1920), description of the color phenomena due to the
action of strong sulphuric acid on egg albumin. They observed in many such as cases that a purple color
was produce on the addition of the sulfuric acid. Among protein amino acids, only tryptophan reacted
with glyoxylic acid in the presence of conc. H2SO4 and gave a purple complex. On the other hand, the
Aspartame has no reaction, remained transparent.

In Biuret test, egg and milk turned into violet. The reagents used were CuSO4 and NaOH. Sodium
Hydroxide used to raise the pH of the solution to alkaline level. The complex of Cu2 ions and nitrogen
atoms made the color CuSO4 solution changed into violet. Color changed is dependent on the number of
peptide bonds in the solution, the more protein, the more pigmented the color change. The test showed
the presence of proteins in the following food. Moreover, the aspartame remained colorless.

The Ninhydrin test results were accurate. The milk solution and egg albumin changed color into
purple. These were because in pH range 4 to 8, all amino acid reacted with ninhydrin, a powerful
oxidizing agent gave a purple outcome. Hence, proteins were present. Aspartame gave a crystal, clear
blue outcome.

During the Sakaguchi test, a red-colored complex formed in both the egg and the milk. The
aspartame produced a shade of light green. Sakaguchi test is specific for Arginine. The test is positive for
containing the guanidine group in Arginine. Guanidine group present in the amino acid reacted with α-
Napthol and alkaline hypobromite which gave a red-colored complex. Hence, proteins containing
Arginine were present.

In Xanthoproteic test, orange-colored and yellowish cloud color was observed in both the egg
and milk. The changed in color was due to the high temperature of which the Phenylalanine, tyrosine
and tryptophan reacted with the conc. HNO3, formed nitro-compounds which were yellow in color, it
turned orange-colored in an alkaline medium. Furthermore, amino acids containing the benzene ring
(aromatic amino acids) were present.
V. CONCLUSION

In conclusion, the students were able to attest the tests and detected that proteins were
present in each: Hopkins-Cole, Biuret, Ninhydrin, Sakaguchi, and Xanthoproteic. Also, the students were
able to determine specific amino acids in protein and the different types of proteins.

VII. RECOMMENDATION

This recommendation is intended for those who will conduct this experiment. This type of
experiment is about identifying proteins, to have further knowledge, the group recommends to have a
research ahead of the possible results of the Biuret Test, Ninhydrin Test, Hopkins Cole Test, Sakaguchi
Test and Xanthoproteic Test. Researching ahead of time can give an idea of the expected result.

VIII. REFERENCES

Elhakim, A. (2010). Protein Denaturation. Retrieved on 09, 06, 2019. Retrieved from
https://www.scribd.com/document/45778524/Protein-Denaturation

Faculty of Medicine, Biochemistry Department. (n.d). Biochemistry Practical: Proteins & Amino Acids
Identification. Retrieved from
https://www.google.com/url?sa=t&source=web&rct=j&url=http://www.magdyelnashar.com/ne
w/images/pdf/Practical.Proteins.and.Amino.Acids.Identification.pdf&ved=2ahUKEwim89LX_Lnk
AhXWMt4KHfIgD7UQFjAKegQIChAB&usg=AOvVaw3AvYxzgWfhNXj5s2ax6gJz

Fearon, W. R. (1920). A Study of some Biochemical Test. No. 2: The Adamkiewicz Protein Reaction. The
Mechanism of the Hopkins-Cole Test for Tryptophan. A New Color Test for Glyoxylic Acid.
Biochemical Journal, 14(5), 548

Izneera P. (n.d). DOC Lab Report. Experiment B: Qualitative Test for Proteins. Retrieved from
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.academia.edu/361539
23/Lab_report&ved=2ahUKEwjO27njtLfkAhXF7WEKHRTLAEUQFjADegQIARAB&usg=AOvVaw3Q
Cnyl36OMsuBAMkogbenK

Kauzmann, W. (1959). Some factors in the interpretation of protein denaturation. In Advance in protein
chemistry (Vol. 14, pp. 1-63). Academic Press.