MBARARA UNIVERSITY OF SCIENCE AND TECHINOLOGY

FACULTY OF MEDICINE

DEPARTMENT OF BIOCHEMISTY

COURSE UNIT: INTRODUCTION TO BIOCHEMISTRY (BCH1111)

Name: NABIRYE MERCY

Registration Number: 2023/MBR/029

GROUP MEMBERS

NAME Registration Number

Nansamba Angel Fortune 2023/MBR/031

Namugema Phoeba 2023/MBR/030

Musinguzi Kenneth 2023/MBR/027

Mutebi Isaac 2023/MBR/028

Nabirye Mercy 2023/MBR/027

Supervisors 1. Mr. Muhereza Deneth

2. Mr.Mutekanya Emmanuel

3. Mr.Mukundane Victor
Abstract.
This experiment was carried out to identify and establish the constituents of the
unknown samples P, Q, R, S, T and U. Various test solutions and reagents were
provided. The experiment was done in a series of steps to identify the constituent
protein or amino acid in the different test solutions basing on the chemical reactions
of the sample with the reagent(s) added. For every step, a single test was carried
out and a sample whose composition was correctly identified or confirmed was
excluded from the subsequent tests which involved further analysis of the
remaining test samples using other reagents. This was done until the identity of all
the samples was ascertained.

Introduction.
Proteins are among the most abundant molecules in living systems and are way
more diverse in structure and function than other macro molecules. Although their
structures and functions vary greatly, all proteins are made up of one or more
chains of amino acids. The amino acids are the building blocks/monomers of
proteins. Amino acids share a basic structure consisting of a central (alpha) carbon
atom bonded to an amino group (NH2), a carboxyl group (COOH), and a hydrogen
atom. Every amino acid also has another atom or group of atoms bonded to alpha
carbon atom, known as the R group which determines the identity of the amino
acid. In formation of proteins, amino acids undergo condensation reaction forming a
peptide bond. The protein formed consists of a free amino group, free carboxyl
group and most importantly R groups that react uniquely with different reagents
giving different colour changes and observations

When subjected to Ninhydrin, the amino acid undergoes oxidative deamination

releasing carbon dioxide, ammonia and aldehyde to produce Hydrindantin. The
ammonia then interacts with another molecule of Ninhydrin to generate
diketohydrin also known as Ruhemann’s complex. This is responsible for strong blue
colour. Using this method, it is possible to identify proteins and amino acids from
non-proteins and non- amino acid compounds.

For the Biuret test, the cupric(Cu2+)in Biuret reagent bind to nitrogen atoms in
peptide bond of proteins forming a violet coloured copper coordination complex.
This reaction occurs in alkaline environment.The formation of purple colour
indicates presence of peptide bonds in the sample and intensity of purple colour is
directly proportional to the concentration of peptide bonds present. For non-protein
compounds and free amino acids the reaction gives negative results.

The Thiol test is used to test for presence of sulphur atoms present in cysteine
residues. When potassium hydroxide is boiled with cysteine, the sulphur present in
cysteine is converted to inorganic potassium sulphide which then reacts with lead
acetate to form a black precipitate of lead sulphide. Hydrolysis of cysteine using
potassium hydroxide is necessary because lead acetate tests for presence of
sulfhydryl group.

The Adamkien reaction is used to detect the presence of tryptophan in a sample as

free amino acid or in a protein structure. The indole side chain in tryptophan reacts
with dilute formaldehyde in presence of concentrated Sulphuric acid forming a
yellow-brown ring.

Xanthoproteic test is a biochemical test used for detection of indolic or phenolic

groups (aromatic amino acids such as tyrosine, tryptophan and phenylalanine).The
aromatic groups in the amino acids are nitrated by heating with concentrated Nitric
Acid to yield intensely yellow-colored nitro derivative. On the addition of alkali, the
residue turns orange due to formation of a salt of tautomeric form of nitro
compound.

Objectives
To determine the identity of the given samples.

Materials
 Ninhydrin reagent 1%  Sodium hydroxide solution
 Biuret’s reagent  Sample P,Q,R,S,T,U
 40% Potassium Hydroxide  Test tubes
 10% Lead Acetate  Pipette filler
 Dilute Formaldehyde  Pasteur pipette
 Concentrated Sulpuric acid  Water bath
 Nitric acid

METHODS AND PROCEDURES USEDS

Ninhydrin test;

0.5mls of samples were pipetted into respective test tubes followed by 2ml of
Ninhydrin reagent. The tubes were placed in a bath and boiled for 2 minutes after
which the colour changes were noted and recorded. The sample which gave
negative results was excluded for the other tests.

Biuret test;

0.5mls of the remaining samples were pipetted into test tubes, and then 2ml of
Biuret reagent added. The test tubes were left to stand for 2 minutes after which
colour changes were noted and recorded. The sample with positive results was
excluded from the processed tests.

Thiol test;

1ml of each of the remaining samples was pipetted into test tubes along with 1ml of
40% potassium hydroxide. 4 drops of 10% lead acetate were added to each test
tube after which the tubes were boiled for 2 minutes and the colour changes
recorded. The test-tube with positive results was again eliminated for the next
processing tests.

Adamkien test;

0.5ml of each of the test solutions was pipetted into test tubes followed by 4 drops
of diluted Formaldehyde and then 1ml of concentrated Sulphuric acid. The test
tubes were then left to stand for 5minutes and the colour changes observed. Again
the sample with positive results was eliminated.

Xanthroproteic test;

1ml of the samples, that remained were then pipetted into respective test tubes,
added 0.5ml of Nitric acid and boiled for 2 minutes. After 2 minutes of cooling, 4ml
of 10% sodium hydroxide were added to each tube and the colour changes
recorded.

TABLES OF RESULTS

Ninhydrin test
Test tube Observation Deduction
P blue solution Protein or amino acid
Q Purple solution Protein or amino acid
R Red color formed Protein or amino acid present
S Blue color formed Protein or amino acid present
T blue color formed Protein or amino acid present
U No color change Non-amino acid
Biuret test
Test tube Observation Deduction
P Blue color Free amino acid present
Q Blue color formed Free amino acid present.

R Black color Free amino acid present.

S Black color Free amino acid present.
T Purple color Albumin present
Thiol test

Test tube Observation Deduction

P Colourless solution Thiol group absent
Q Colourless solution Thiol group absent
R Black color Cysteine present
S Colourless solution Thiol group absent
Adamkien test

Test tube Observation Deduction

P Colourless Aliphatic amino acid
Q Colourless Aliphatic amino acid
S Yellow-brown ring Tryptophan present

Xanthroproteic Test

Test tube Observation Deduction

P Colourless solution Alanine present
Q Orange coloration Tyrosine present
Discussion of results.

In the Ninhydrin test, ability of Ninhydrin (2, 2-dihydroxyindane-1, 3-dione) to react

with ammonia and amines (primary and secondary) was used find out which tubes
contain amino acids or proteins. Reaction of free amino acids produced carbon
dioxide, an aldehyde with one less carbon atom than the original amino acid and
Hydrindantin which reacted normally with ammonia to give “Ruhemann’s complex”
purple or blue in colour. Hydrindantin gave positive results for samples; P, Q, R, S, T
indicating presence of amino acids or proteins and a negative result for sample U
thus a non-protein.

Cupric ions react with nitrogen atoms involved in peptide bonds, displacing peptide
hydrogen atoms under alkaline conditions; forming tri/tetra dentate chelation to
produce the "biuret" colour (purple).This however is not possible with the free
amino acids which have no peptide bonds. The tube with the sample T tested
positive showing presence of albumin.

In the Thiol test, the Thiol group (─SH) of Cysteine reacted with lead acetate giving
lead sulphide (a black precipitate) as seen for sample R. In the Biuret test, proteins
form complexes with copper (Cu2+) in alkaline solution, purple in colour.
In the Adamkien test, tryptophan was condensed with dilute formaldehyde in
presence of concentrated Sulphuric acid forming a yellow-brown ring which was
positive in sample S.

The Xanthroproteic Acid Test targeted aromatic amino acid side chains for tyrosine
and tryptophan. Nitration of the aromatic ring by concentrated nitric acid produced
a nitro compound (Xanthroproteic acid) that gave an orange colour as a positive
indicator. This was seen in sample Q, and identified it as Tyrosine, leaving sample P
as Alanine.

Conclusion.

Sample P contained alanine, sample Q contained tyrosine, sample R contained

cysteine, sample S contained tryptophan, Sample T was the protein (albumin) and
sample U was a non-protein.

References

"biuret test principle." microbenote.com. n.d. microbenote.com.

David L Nelson, Michael M Cox. Lehniniger Principles of Biochemistry. n.d.

principles and techniques of biochemistry and molecular biology. n.d.

https://ghr.nlm.nih.gov/primer/howgeneswork/protein

Students’ Biochemistry Practical Handbook (2018), Department of Biochemistry,

Mbarara University of Science and technology

www.khanacademy.org

http://www.toppr.com/guides/chemistry/ninhydrin-test/

www.microbenotes.com