UNIVERSITY OF GHANA, LEGON

DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY

EXPERIMENT 12

DETERMINING THE PROPERTIES OF PROTEINS AND AMINO ACIDS

USING QUALITATIVE ANALYSIS

JOSEPH ARMAH BAIDOO

ID: 10852150

NOVEMBER 5, 2021

GROUP 15

MABEL APPIAH- ADJEI

KUUMAZAH BLESS KWAKU

ANAAFI- KWAPONG CHRISTIAN

MARFO SAMUEL

KWAYISI- DARKWAH CALEB KORANTENG

ISHAAQ AGBERE NAJMU- DEEN

OWUSU EMMANUEL DONKOR

JOSEPH BAIDOO

1
INTRODUCTION

Proteins belong to the four essential macromolecules present in all life forms; the

others being carbohydrates, lipids and nucleic acids (Barbour et al, 1929). They are

the most abundant biomolecules present and can occur in many forms including

enzymes, antibodies, hormones and components of the cell skeleton including the cell

wall. Proteins are obtained from animal products even though they can also be found

in plant products such as legumes and nuts (Zumdahl &Zumdahl, 2007). In the body,

proteins make about 15% of the body’s weight (Berg et al, 2002). Proteins are very

essential molecules in that life would be very difficult without them. In the body,

proteins are responsible for repairing worn out tissues, catalyzing reactions in the

form of enzymes, sending signals as hormones and also fighting pathogens as B cells

(Kriz et al, 2009). Primarily, proteins store genetic information encoded in DNA

sequences in the nucleus (Van Der Vies, 1953). Medically proteins are used in

making drugs and antibiotics to fight diseases. It is estimated that about 10,000

different proteins are present in the human body (Bhatia et al, 2015).

Like carbohydrates, proteins are made of building blocks known as amino acids (Kotz

et al, 2008). Amino acids are chemicals composed of carbon, hydrogen, oxygen,

nitrogen and traces of sulphur in some (Klein et al, 2015). The amino acids form

bipolymer proteins constructed from end to end and display a wide range of functions

and structures. All amino acids have a common structure. All amino acids have a

common backbone of a central carbon bonded to an amino group, a carboxyl group, a

hydrogen atom and a side chain which is specific to different amino acids (Boyer,

2000). Hence, the simplest amino acid will have a hydrogen atom as side chain and

this amino acid is known as glycine. Due to their different side chains, amino acids

can be grouped into four main groups; polar (histidine), non polar (leucine), acidic

2
( aspartate) and basic (arginine).

The nature of the amino acid chain determines the overall structure and reactivity of

the molecule. Due to this, some amino acids bind covalently, ionically or through

disulphide bonds (Harris, 2007). Proteins found in nature are polypeptides of twenty

main amino acids of which the body is able to produce about half and the other half

has to be obtained through feeding (Chichester et al, 1972). Hence they are known as

essential amino acids. Peptide linkages are formed when the amino group of one

amino acid reacts with the carboxyl group of the other amino acid. Depending on the

number of amino acids, the result can be a dipeptide, tripeptide and many more (Cox

et al, 2000). When this happens, proteins are formed and hence proteins are called

polypeptides of amino acids. Proteins can also be formed from many polypeptide

chains.

Amino acids and proteins are able to undergo different reactions which are entirely

based on special properties they possess (Galewska et al, 2013). Due to similar

properties of proteins and amino acids, different results are obtained when they are

reacted with different reagents. Hence, the reactions of proteins and amino acids can

be used to identify the specific group or property, and hence amino acid or protein one

is dealing with (Davis et al, 2000) .

This experiment sought to use various reagents to qualitatively identify specific side

group structures and chains in amino acids, composition of some proteins and also the

chemical content of an unknown substance.

3
MATERIALS

Millon’s reagent, concentrated nitric acid, concentrated sodium hydroxide, Biuret’s

reagent, ninhydrin, 20% sodium hydroxide, lead acetate, albumen, tyrosine, glycine,

an unknown sample, gelatin, tryptophan, cystine, cystein, proline, methionine ,

boiling water bath and water

METHOD

Samples of amino acids and proteins were provided for this experiment. Different

qualitative tests were carried out for each test and the results were recorded. The first

test done was the Millon’s test. In this test the samples used were albumen, tyrosine,

glycine and an unknown sample. About 3 ml of each test sample were collected into

three separate and labeled test tubes. 5 drops of the Millon’s reagent were then added

to each sample in the test tube and the results were recorded. After the addition of the

reagent, the samples were brought to be heated carefully over a boiling water bath. All

observations including colour changes were all noted for this experiment.

In the next test, tyrosine, gelatine, albumen, glycine, tryptophan and the unknown

were subjected to the Xanthoproetic test. In this test, about 3 ml of the listed samples

were collected into six different test tubes which were labeled. 1 ml of concentrated

nitric acid wad added to each sample in the test tube. The resulting solution was then

brought to the boiling water bath to be heated. Heating was done for about five

minutes. The samples were removed from the bath and allowed to cool by running

water around the test tubes. After cooling, drops of concentrated sodium hydroxide

were added carefully to the mixtures until the respective solutions turned alkaline. All

observations were recorded.

4
In the following test, the Biuret test was performed on albumin, tyrosine and glycine.

For this test, 1 ml of the albumin, tyrosine and glycine were sampled into three

labeled and separate test tubes. 4 ml of the Biuret reagent was added to the samples.

Each mixture was mixed very well and left to stand for about 30 minutes. Meanwhile,

a control was set up using water. The observations including the colour changes of the

samples were all recorded.

In the Ninhydrin test, 0.5 ml 0.1% ninhydrin was added to 2 ml of samples. The

resulting mixtures were heated in a boiling water bath for 15 - 30 minutes. Eight

samples were used, hence this part required eight test tubes with each labeled with the

sample to be tested in it. The samples used were tyrosine, gelatin, albumen, glycine,

tryptophan, cystine, cystein and proline. Some of the samples changed colors and all

the observations were recorded.

The last test performed was the Sulphur test. In this part, cystine, cystein and

methionine were the samples used. 1 ml of each of the samples were collected into

three different test tubes with each labeled. A

bout 1 ml of 20% NaOH were added to each test tube. The resulting mixture were

boiled for about five minutes. When the solutions were removed, the samples were

allowed to cool. A drop of basic lead acetate was added to each mixture in the test

tubes. The observations for this test were all recorded.

5
RESULTS

Careful procedures and accurate timings were ensured in order to produce accurate

results. Table 1 below shows the test and the samples used with their corresponding

inferences obtained. From the table, it is seen that only tyrosine gave a positive test by

turning red for the Millon’s test. Glycine and the unknown both recorded no color

change and hence the negative test. A yellow colour formed in albumen which

indicates a negative test. Also from the table, it can be noted that all the samples used

in the xanthoproteic test gave positive result except glycine and gelatin. Both samples

did not give any colour change while the rest turned yellow.

In the Buiret test, it can be noted that the albumen was the only sample to give a

positive test by producing a violet colouration. Tyrosine gave a fairly pale blue colour

which is a negative test. Glycine and the control ( water) both gave a pale blue color

which also indicated a negative test. Tyrosine, albumen, glycine and proline were the

only ones to give a positive test for the ninhydrin test by giving a purple colouration

except proline which gave a yellow colouration. Gelatin and cytine did not change

colour, hence the negative test. Tryptophan and cystein both produced brown colours

which also indicates a negative test. In the last test, cystein and cystine recorded dark

brown colors which are positive tests for sulphur. Methionine did not change colour.

Hence it gave a negative test.

6
TABLE 1: OBSERVATIONS RECORDED FROM THE DIFFERENT TESTS

ON THE VARIOUS TEST SAMPLES AND THEIR INFERENCES

TEST SAMPLE OBSERVATION INFERENCE

Albumen Yellow colour formed Negative test

Tyrosine Red colour formed Positive test

MILLON’S Glycine No colour change Negative test

Unknown No colour change Negative test

Tyrosine Deepening of yellow colour Positive test

Gelatin No colour change Negative test

Albumen Deepening of yellow colour Positive test

Xanthoproteic Glycine No colour change Negative test

Tryptophan Deepening of orange colour Positive test

Unknown Deepening of yellow colour Positive test

Albumen Violet colour Positive test

Tyrosine Pale blue colour Negative test

Buiret Glycine Pale blue colour Negative test

Water Pale blue colour Negative test

Tyrosine Purple colour Positive test

Gelatin No colour change Negative test

Ninhydrin Albumen Purple colour Positive test

Glycine Deep purple colour Positive test

Tryptophan Brown Negative test

7
 Cystine No colour change Negative test

Cystein Light brown colour Negative test

Ninhydrin Proline Yellow colour Positive test

Unknown No colour change Negative test

Cystine Dark brown colour Positive test

Samples Cystein Dark brown colour Positive test

Methionine No colour change Negative test

Table 1 shows the tests performed for the various samples and the observations

recorded with their corresponding inferences.

Figure 1 showing the images of the Millon’s test for the three solutions used.

From left to right are the samples glycine, unknown, tyrosine and albumen.

8
Figure 2 showing the images of the Xanthoproteic test for all six samples used.

From top left to the bottom right are the samples in order gelatin, tyrosine, albumen,

glycine, tryptophan and the unknown.

Figure 3 showing the images for the sulphut test for three samples

Samples are in order cystein, cystine and methionine

9
Figure 4 showing the images for the Buiret test results for four samples.

The samples are in order glycine, water, albumen and tyrosine.

Figure 5 showing the images for the Ninhydrin test results for the nine samples

used

Samples are in the order proline, albumen, tyrosine, tryptophan, cystein, glycine,

gelatin, cystine and unknown.

10
DISCUSSION

Proteins are able to undergo many different reactions due to their different functional

groups present in their molecules. Hence, if a particular group of proteins or amino

acids share a common functional group, they are likely to have similar reactions with

the same reagents and this is shown by the colour they produce from these reactions

(Zumdahl &Zumdahl, 2007).

Millon’s test is used to identify amino acids with a phenol group attached to the

molecule. A phenol group has a hydroxyl group attached to a benzene ring. When a

phenol group comes in contact with the Millon’s reagent, it gets nitrified, causing it to

form a complex with mercury in the reagent (Bhatia et al, 2015). This complex gives

the characteristic red colour for a positive test. Hence, an amino acid with a phenol in

it will give a positive test. From the experiment, only tyrosine gave a positive test

among the samples used. This shows that tyrosine has a phenol side chain. Albumen

is a protein, hence the negative test (Klein et al, 2015). Glycine does not have a

phenol group, hence it was expected to give a negative test. The unknown molecule

tested negative for the test, hence it can be explicitly concluded that it does not

possess a phenol group.

Xanthoproteic test are used to identify aromatic compounds of amino acids and

proteins. When nitric acid is added to an amino acid with an aromatic side chain, it

reacts with the aromatic side chain to produce a yellow or orange colour. Upon the

addition of alkali the yellow or orange colour deepens(Davis et al, 2000). From the

experiment, tyrosine, albumen, tryptophan and the unknown sample all gave positive

test. This is expected of tryptophan, tyrosine and the albumen since they all have

aromatic structures in them. It can also be concluded that the unknown has aromatic

11
side chain since it also tested positive. However, gelatin and glycine gave negative

results indicating that they do not have an aromatic side chain.

The Buiret test is a general test used to identify all proteins. This is based on the

principle that proteins are formed by peptide bond (Davis et al, 2000). Upon addition

of the buiret reagent the peptide bonds in them break to form smaller sub units giving

the violet pigment. Albumen is a protein usually found in egg. When tested with the

Buiret reagent, it gave a positive test as expected (Galewska et al, 2013). Tyrosine

and glycine gave negative results as they are not proteins but rather amino acids. The

water control set up also gave a negative test as it does not contain proteins.

Ninhydrin test is a test used to identify proteins or amino acids with primary or

secondary amines in them. Ninhydrine reacts with amines ( primary and secondary)

to give Ruhemann’s solution which is purple in colour (Van Der Vies, 1953). The

purple colour is what is observed as a positive test. Except for proline which gives a

yellow colour as a positive test, all other amino acids will give a purple or violet

colour as positive test. This is because proline possesses a secondary amine while the

other amino acids have primary amines (Galewska et al, 2013). Tyrosine, glycine,

albumen and proline were the only ones to give a positive test due to primary and

secondary amines in them. Gelatin, tryptophan, cystine, cystein and the unknown

gave negative results because they do not have primary or secondary amines in them

(Chichester et al, 1972).

The sulphur test is to identify the presence of sulphur in amino acids. Sulphur breaks

of from the side chain of its amino acid in the presence of a base and lead acetate. A

positive test will be a black precipitate of sulphur which will be released (Chichester

et al, 1972). Cysteine and cystine both contain thiol groups (-SH), hence they gave a

12
positive test by giving a black solution (Klein et al, 2015). Methionine has a sulphur

in its structure. However the sulphur does not break off in the presence of an alkali,

hence it will not give a positive test. From the experiment, methionine gave a negative

test as expected.

CONCLUSION

The experiment conducted has proven that major groups and side chains of amino

acids are responsible for their reactivity. Hence a group of amino acids with similar

groups will have similar reactions (Galewska et al, 2013). Examples of such groups

are the phenol group, thiol groups, aromatic groups and polypeptide chains as present

in proteins. From the data collected, it can be concluded that the albumen is a

molecule that contains an aromatic group like that in tyrosine (Galewska et al, 2013).

Also, the albumen is a protein that has a primary amine structure in its chemical

formula. Gelatin does not possess any of these groups in its structure since it gave

negative tests to all the tests.. Finally it can be concluded that the unknown sample

contains an aromatic group in its structure. It does not have a phenol in its structure

and it does not also have a primary or secondary amine in its side chain. Due to this

structure, the unknown is likely to be phenylalanine.

13
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Harcourt

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