Activity No.

AMINO ACIDS AND PROTEINS

INTRODUCTION

Proteins are Complex nitrogenous organic substances involved in all phases of chemical and physical
activity in the living cells. They contain 16% nitrogen and sometimes sulphur and phosphorus. All living
cells are primarily composed of proteins and are compose of chains of AMINO ACIDS. The richest sources
of protein in food are lean meat, cheese, and eggs.

Proteins are polymers of α-amino acids with 20 common amino acids: 9 Nonpolar amino acids, 6
Polar-neutral amino acids, 2 acidic amino acids, and 3 basic amino acids. Proteins are also classified as
SIMPLE (contains simple amino acids) and CONJUGATED (protein + nonprotein moiety) based on their
composition. Example of simple and conjugated amino acids are shown below.

Structure of α-amino acids

Serum Albumin – simple protein Hemoglobin – Conjugated Protein

REACTIVITY OF AMINO ACIDS AND PROTEINS

COLOR REACTIONS

Millon’s Test

Indicates the presence of hydroxyphenyl groups in proteins that is present in Tyrosine and amino acids containing
this group. Presence of hydroxyphenol is detected by the formation of a coloured-mercury complex (shown below) with
hydroxyphenol producing a white precipitate that turns red when heated.

Xanthoproteic Test

Also known known as the Yellow Protein Reaction which is based on the nitration of aromatic rings (shown below) of
the amino acid such as tyrosine, phenylalanine, and tryptophan producing yellow precipitate and turns orange upon
addition of an alkali.
Biuret Test

Test to indicate the presence of peptide bonds by coordination of the nitrogen and oxygen lone-pairs of the peptide
bond and water, respectively, with the cupric ions producing purple to violet coloured complex.

Sulfur Test

This test indicates the presence of sulphur groups present in the amino acid chain such as methionine, cysteine, and
cysteine by precipitation of hydrolysed protein with lead acetate with subsequent formation of a black or brown lead
sulphide precipitate.

Molisch Test

Test for the presence of carbohydrates via dehydration with a strong acid to produce 5-hydroxymethylfurfural which
then further reacts with α-naphthol to form a purple-coloured dye product.

Hopkin’s Cole Test

Known as the glycoxylic acid reaction, is a test used for the detection of tryptophan in proteins. It is done by the
addition of glycoxylic or glacial acetic acid solution and further addition of sulphuric acid. A purple interface is formed
when tryptophan is present.

Heller’s Ring Test

Test to indicate that denaturation of proteins can be carried by strong acids. the test uses concentrated Nitric acid
forming two layers. Formation of a white precipitate in the interface appears signifying a positive result. This test is
also used for detection of proteins in urine.

Precipitation Reaction

Proteins are separated or precipitated using different methods such as Heating, Salting Out effect, use of Acids,
Alcohol, Organic Solvents, use of positive ions of heavy metals such as Pb2+, Zn2+, Cu2+, Cd2+, Hg2+, and Fe3+, and
use of Alkaloidal reagents producing insoluble proteinate.

GENERAL OBJECTIVES

At the end of the experiment, the students should be able to:

1. identify various chemical components of proteins;
2. conduct chemical reactions and tests of amino acids; and
3. explain the significance of each test.

CHEMICALS AND METARIALS

Chemicals Materials

Prepared by each group from expt. # 3 Test tubes 40 pcs

1% albumin solution (10mL) 10 pcs
1% Casein solution Test tube 2 pcs
cover 1 pc
Provided by Lab technicians Test tube 1 set
Standard Albumin Solution holder 1 pc
Standard Casein Solution Test tube 1 unit
10% NaOH rack 1 pc
0.1% CuSO4 Water bath
Conc. Ammonium hydroxide Wash bottle
Millon’s reagent Hot plate
Gelatin Thermomete
Glacial Acetic acid or Glycoxylic acid r
Conc. Sulfuric acid
Saturated Lead acetate
Conc. Nitric Acid
Dilute Acetic Acid
Dilute NaOH
95% Ethanol
Solid NaCl
Solid ammonium sulfate
 5% tannic acid
Saturated picric acid
5% potassium ferrocyanide
Dilut
e HCl
pH
paper
PROCEDURE

A. Colour Reactions of Proteins (use standard Std. Albumin and Std. Casein only)

Millon’s Test
1. Place 0.5mL of protein sol’n. into a 10ml test tube.
2. Add 5 drops of Millon’s reagent and heat in a boiling water bath.
3. Record Result

Xanthoproteic Test
1. Place 1mL of protein sol’n. into a 10mL test tube.
2. Add 1mL of conc. HNO3 into the solution.
3. Heat the mixture for about 3 minutes at 70oC and allow to cool at room temperature.
4. Slowly add 10% NaOH drop by drop until the mixture becomes alkaline.
5. Note the change in color and record results.

Biuret Test
1. place 0.5mL of protein sol’n. into a 10mL test tube.
2. Add 5 drops of 10% NaOH and 5 drops 0.1% Copper Sulfate and Shake.
3. Observe colour and record results.

Sulfur Test
1. Place 0.5ml of protein sol’n. into a 10ml test tube.
2. Add 5 drops of 10% NaOh and 1ml of saturated Lead Acetate.
3. Heat in a boiling water bath.
4. Record Results.

Molisch’s Test
1. Place 2 mL of protein sol’n. into a 10mL test tube.
2. Add 1 drop of Molisch’s reagent
3. Pour 1-2 mL of conc. H 2SO4 down the side of the test tube, so that it forms a layer at the bottom of the
tube.
4. Observe color of the interface.
5. Record results.

Hopkin’s Cole Test

1. Place 1mL of protein suspension in a 10-mL Test tube.
2. Add 1mL of glacial acetic acid or glycoxylic acid sol’n. Mix
3. Carefully add 1mL of conc. H2SO4 down the side of the tube in order to form an interface. Let stand for 5
minutes.
4. Record the colour of the interface.

Heller’s Ring Test

1. Place 1mL of protein solution (Albumin) In a 10-mL Test tube.
2. Carefully add 1mL of conc. Nitric Acid down the side of the tube in order to form an interface. Let stand
for 5 minutes.
3. Record the colour of the interface.

B. Precipitation of Proteins (Denaturation)

Effects of Heat
1. Place 1ml of protein solutions into three 10-mL test tubes labelled 1, 2, and 3.
2. Add 2 drops of acetic acid in test tube 1, 2 drops of 10% NaOH in test tube 2. Test tube 3 serves as
control.
3. Gradually heat test tubes in a water bath and monitor the temperature using a thermometer.
4. Record the temperatures when coagulation of protein takes place in each test tube.
5. Record results.
 Coagulation Test
1. Place 2ml of protein solutions in to a 10-mL test tube and heat to boiling.
2. Repeat procedure using gelatin as sample.
3. Record precipitate formed.

Effect of Alcohol.
1. Place 1mL of protein solutions into a 10-mL test tube.
2. Add 1mL of 95% ethanol.
3. Record results.

Neutral Salts
1. Place 2mL of protein sol’ns. into two 10-mL test tube labelled 1 and 2.
2. Add excessive amount of NaCl Sol’n. in test tube 1 and (NH 4)2SO4 sol’n. in test tube 2.
3. Record Results.

Precipitation of Heavy Metal Ions

1. Place 1mL of protein sol’ns. into three 10-mL test tubes labelled 1, 2, and 3.
2. Determine pH using pH strip. Record the pH.
3. If pH<6, add 1 drop of 10% NaOH until pH>6.
4. If pH>6, add 3 drops of 1% mercuric chloride, 1% silver nitrate, and 1% lead acetate to test tubes 1, 2,
and 3, respectively.
5. Record Results.

Precipitation with Alkaloidal Reagents

1. Place 1mL of protein sol’ns. into three 10-mL test tubes labelled 1, 2, and 3.
2. Add 2 drops of 5% tannic acid, saturated pieric acid sol’n., and 5% potassium ferrocyanide into test tubes
1, 2, and 3, respectively.
3. Make sure that solutions have pH<4, if not then add 1 drop of dilute HCl.
4. Observe precipitate formation and Record Results.

References

1. Seager, S.L., Slabaugh, M.R.; Saftey-Scale Laboratory Experiments for Chemistry for Today 7 th edition, Brooks/Cole Cengage Learning, 2011.
2. Walsh, Edward O'Farrell (1961). An Introduction to Biochemistry. London: The English Universities Press Ltd. pp. 406–407. OCLC 421450365.
3. Chatterjea (1 January 2004). Textbook of Biochemistry for Dental/Nursing/Pharmacy Students. Jaypee Brothers Publishers. p. 51. ISBN 978-81-8061-204-6.
4. P. M. Swamy (2008). Laboratory Manual on Biotechnology. Rastogi Publications. p. 90. ISBN 978-81-7133-918-1.
5. Elizabeth A. Martin (ed) (25 February 2010). Concise Colour Medical Dictionary. Oxford University Press. p. 335. ISBN 0-19-955715-2.

Group No.: 12 Date Performed:___________________

Name: ____________________________ Date Submitted:___________________

Instructor: Nesteve John B. Agosto, RCh, MSc

 Score: ___________/45 pts
Rating: __________

Activity No. 7

AMINO ACIDS AND PROTEINS

I. OBSERVATIONS/DATA
A. COLOR REACTIONS OF PROTEINS (2pts each)
Observable Result
Chemical Test
Std. Albumin Isolated Albumin Std. Casein Isolated Casein
Millons Test
Xanthoproteic Test Light-yellow
Clear solution with
colored solution
a light-yellow ring
with yellow stain
on skin
Biuret Test Purple colored Purple colored Light-purple Very Light-purple
solution solution colored solution colored solution
Sulfur Test

Color of the interface

Chemical Test
Std. Albumin Prepared Albumin Std. Casein Prepared Casein
Molisch’s Test Cloudy white Cloudy white Cloudy white Cloudy Light-
interface interface interface brown interface
Hopkin’s Cole Test
Heller’s Ring Test Yellow-green ring White-yellow ring

B. PRECIPITATION OF PROTEINS (2 pts each)

a. Effects of Heat
Temperature of Coagulation

Std. Albumin Std. Casein

Test tube 1

Test tube 2

Test tube 3

b. Coagulation Test
Samples Observable Results
Std. Albumin Formation of a thick white coagulum
Std. Casein Formation of a white precipitate
Gelatin Do not coagulate on heating/No precipitate formed

c. Effects of Alcohol
Samples Observable Results
Std. Albumin White solution with a small white precipitate at the bottom part
Std. Casein Clear solution with a small white precipitate at the bottom part

d. Neutral Salt
Observable Results

Std. Albumin Std. Casein

Test tube 1 Formation of small precipitate No precipitate formed

Test tube 2 Formation of small precipitate No precipitate formed
e. Precipitation using Heavy Metals

Observable Results

Std. Albumin Std. Casein

Test tube 1

Test tube 2

Test tube 3

f. Precipitation with Alkaloid Reagents

Observable Results

Std. Albumin Std. Casein

Test tube 1 Formation of a neon Formation of a white/light-green

green precipitate precipitate
Test tube 2 Cloudy green solution Neon green solution/No precipitate
formed
Test tube 3

III. Answers to Questions

1. What chemical test must be used to detect the presence of the following amino acids in a protein sequence? (1
pt each)

a. Tryptophan - Xanthoproteic Test/Hopkin’s Cole Test

b. Cystein - Nitroprusside Test/Sulfur Test
c. Phenylalanine - Xanthoproteic Test

2. What simple test can be used to detect proteins in a solution? (2 pts)

The Biuret test, it is a chemical test used for detecting the presence of peptide bonds. In the presence of
peptides, a copper (II) ion forms violet-coloured coordination complexes in an alkaline solution. The Biuret reaction
can be used to assess the concentration of proteins because peptide bonds occur with the same frequency per amino
acid in the peptide.