Biochemistry (BTBIMOLAB) 1

Laboratory Manual

Laboratory Procedure
Proteins and Amino Acids Activity
5

INTRODUCTION

Proteins are the major constituents in every living cell. They have a variety of functions.
They catalyze biochemical reactions, regulate the activity of various organs in the body,
counteract the adverse effects of antigens, transport molecular oxygen, and serve as
structural materials of the muscle, skin, and hair.

Chemically, proteins are macromolecules that contain alpha amino acids as the building
blocks. These amino acids are joined together by the peptide bond, also called peptide
linkages.

A. Amino Acids

All amino acids are carboxylic acids that include an amino (NH2) functional group
attached to C-2 (the alpha carbon) and are thus called –amino acids.

The structure also includes another group, R, which is variable depending on the amino
acid. For example, when R is H, the amino acid is glycine; when R is CH 3, the amino
acid is alanine.

B. Polypeptides and Proteins

A peptide linkage is an amide structure is formed by splitting out a molecule of water

between the carboxyl group of one amino acid and the amino group of another. If 40-50
amino acids are linked together, the polymer is called a polypeptide.

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 2
Laboratory Manual

The figure above shows a segment of a polypeptide chain formed from -amino acids.
The R’s represent the side chains (variable groups) of the amino acids and the arrows
point out the peptide linkages in the polypeptide chain.

In a typical protein, hundreds – sometimes thousands – of amino acids are linked

together to form the primary structure of a protein. In the early 1950’s Frederick
Sanger introduced the concept that each kind of protein has a specific amino acid
sequence. He succeeded in determining the amino acid sequence of insulin and was
awarded the Noble Prize for Chemistry in 1958 for his work.

The polypeptide chains of proteins also have a secondary structure or configuration

which occurs when the primary chains are coiled into alpha helices or arranged side-by-
side as pleated sheets which are stabilized by hydrogen bonding between adjacent
amino acids. The final folded shape of protein, referred to as the tertiary structure, is
driven by the chemical nature of its R groups and how they interact with water. Finally,
many tertiary level polypeptides associate together to form a functional protein known
as the quaternary structure.

Because the amino acids in all proteins include an amino group, proteins differ from
carbohydrates and fats not only in their functions in the living organism, but also in
elemental composition. In addition to carbon, hydrogen and oxygen, which are present
in carbohydrates and fats, proteins contain nitrogen. Because some amino acid R groups
include sulfur, sulfur is present in all proteins that include amino acids cystine, cysteine,
and methionine. Additional elements such as phosphorus, iron, copper, zinc, and iodine
also occur in certain complex proteins. These elements are not part of the primary
protein structure but are constituents of non-protein substances combined with proteins.

C. Tests for Color Reactions

The peptide bond of proteins is detected by the Biuret test. Compounds containing two
or more peptide linkages give a characteristic purple color with dilute copper sulfate
solution in alkaline medium. This is due to the coordination of the cupric ions with the
lone pairs of the amide nitrogen and oxygen of water.

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 3
Laboratory Manual

Specific amino acids, whether in the combined form in proteins or in the free state, give
specific color reactions which are used to detect their presence.

The phenolic group in the amino acid tyrosine is detected by the Millon’s test. A white
precipitate is formed which becomes red on heating. The red color is due to the mercury
salt of the nitrated tyrosine.

Aromatic rings in the amino acids such as those of phenylalanine and tyrosine are
readily nitrated. This is the basis of the Xanthoproteic test. A yellow color is produced
which turns orange upon the addition of a base.

When sulfur-containing amino acids are boiled with an alkali, the sulfhydryl or disulfide
groups are converted to an inorganic sulfide, Na 2S. This reacts with lead acetate to form
a black precipitate of PbS. Cysteine and cystines in the free state and in proteins give a
positive result to the sulfur test.

The indole ring of tryptophan condenses with glyoxalic acid in the presence of sulfuric
acid to form a violet-colored complex. This is the basis of the Hopkins-Cole test.

Ninhydrin test is used to detect the presence of the amino group in alpha amino acids.
When amino acids are heated with ninhydrin (triketohydrindenehydrate), ammonia,
carbon dioxide and aldehyde are produced. The NH 3 liberated combines with one mole
of the reduced ninhydrin and one mole of the oxidized ninhydrin to form Ruhemann’s
purple:

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 4
Laboratory Manual

Qualitatively, ninhydrin is used to determine the position of amino acids in

electrophoresis, in paper chromatography, and in thin layer chromatography. It is also
used to estimate the amount of amino acids present. The intensity of the color produced
is related to the concentration of the amino acids.

APPARATUS/MATERIALS CHEMICALS/REAGENTS

Borrow: Request:
Bunsen burner 4 mL 1% albumin
Iron ring 3 mL 1% gelatin
Iron stand 0.5 mL 1% phenol
2 mL 10% sodium hydroxide, NaOH
Bring: 1 mL Hopkins-Cole reagent
Marbles 0.5 mL concentrated ammonium hydroxide,
NH4OH
2 mL concentrated sulfuric acid, H2SO4
0.5 mL 1% glycine
1 mL 1% copper (II) sulfate, CuSO4 (cupric
sulfate)
2 mL 0.1% ninhydrin solution
0.5 mL 0.2% albumin solution
0.5 mL ammonia water
0.5 mL 0.2% urea
0.5 mL 0.2% glycine
1 mL concentrated nitric acid, HNO3
0.2 mL albumin
4 mL 95% ethanol
4 mL 70% ethanol

PROCEDURE

A. Biuret Reaction

SAFETY PRECAUTION:
Always wear safety glasses with side shields, mask, and gloves when
working with chemicals.

1. Place 10 drops each of the following in separate test tubes: 1% albumin and 1%
glycine. Add 5 drops 10% sodium hydroxide and 3 drops 1% copper (II) sulfate
to each test tube. Mix thoroughly and observe the formation of a pink or violet
color. Record your results.

2. Place a pinch of urea in a dry test tube. Heat the test tube over a low flame until
the urea melts (DO NOT CHAR!) and a gas is evolved. Note the odor of the gas

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 5
Laboratory Manual

produced. Cool, dissolve the contents in 20 drops of distilled water, and add 5
drops 10% NaOH and 3 drops 1% CuSO4. Compare the results with number 1 of
this test.
(NOTE: Please refer to page 7 for the Proper Waste Disposal of used samples)

B. Millon’s Test (SEARCH FOR THEORETICAL RESULTS)

1. Into 3 separate test tubes place 20 drops each of the following: 1% albumin, 1%
gelatin, and 1% casein. Add 6 drops of Millon’s reagent to each test tube. Shake
and boil in water bath to the appearance of a red color. Record your observation.

2. Perform the test on 20 drops of 1% phenol. Heat in a water bath if necessary.

Observe. Compare result with number 1 of this test.

C. Xanthoproteic Test

SAFETY PRECAUTION:
When handling solutions with concentrated nitric acid, wear
Safety glasses with side shields, mask, and gloves. Flammable
liquid and vapor, harmful if swallowed, causes severe skin burns and eye damage, and
toxic if inhaled.

1. Into 2 separate test tubes place 10 drops of the following: 1% albumin and 1%
gelatin. Add 1-2 drops of concentrated HNO3 to each test tube. Mix thoroughly.
If the color change is not apparent, add another drop or two. Note the formation
of a heavy white precipitate. Heat in a water bath. Observe the change of
color. Cool and add a few drops of concentrated NH 4OH. Note the change in
color intensity of the substance.

2. Repeat the test with 10 drops of 1% phenol solution. Compare result with
number 1 of this test.
(NOTE: Please refer to page 7 for the Proper Waste Disposal of used samples)

D. Test for Sulfur (SEARCH FOR THEORETICAL RESULTS)

SAFETY PRECAUTION:
Always wear safety glasses with side shields, mask, and gloves
when working with chemicals. It may cause damage to organs
through prolonged or repeated exposure. Harmful if swallowed, can cause skin irritation,
serious eye damage, corrosion, or irritation and very toxic to aquatic life with long
lasting effects. Avoid release to the environment.

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 6
Laboratory Manual

1. To 10 drops of 1% albumin, add 5 drops of 10% NaOH and 2 drops of 5% lead

(II) acetate solution. Shake and boil over a water bath. Observe the formation of
a black precipitate.

2. Repeat the test with a pinch of solid gelatin, and peptone in separate test tubes.
Compare results with number 1 of this test.
(NOTE: Please refer to page 7 for the Proper Waste Disposal of used samples)

E. Hopkins-Cole Reaction
SAFETY PRECAUTION:
Always wear safety glasses with side shields, mask, and gloves
when working with concentrated sulfuric acid (H2SO4) as it may
cause severe skin burns and eye damage.

1. Mix 10 drops of Hopkins-Cole reagent to 10 drops of the following solutions in

separate test tubes: 1% albumin, and 1% gelatin. Incline the test tube and
carefully add 10 drops of concentrated H2SO4 down the side of the test tube. DO
NOT SHAKE. A violet ring formed between the two layers is a positive result.
Record your observations.

F. Ninhydrin Reaction

SAFETY PRECAUTION:
When handling Ninhydrin solution, wear safety glasses with side shields,
mask, and gloves. Causes serious eye irritation and damage.

1. To 10 drops of neutral 0.2% albumin solution add 10 drops of 0.1% freshly

prepared ninhydrin solution. Cover the test tube with a marble and boil over a
water bath. Note the purple color produced.

2. Repeat the test with a. ammonia, b. 0.2% urea, and c. 0.2% glycine instead of
0.2% albumin. Compare the results.
(NOTE: Please refer to page 7 for the Proper Waste Disposal of used samples)

G. Alcohol

SAFETY PRECAUTION:
When handling 95% alcohol, wear safety glasses with side shields,
mask, and gloves. Highly flammable liquid and vapor.
Keep away from heat, sparks, open flames, and hot surfaces. Harmful if swallowed, can
cause skin irritation and serious eye damage, corrosion, or irritation. Avoid breathing
mist, vapors, or spray as it may cause respiratory irritation.

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 7
Laboratory Manual

Place 10 drops of the following test solutions in separate test tubes: 1% albumin
and 1% gelatin. Add 2 ml mL of 95% alcohol to each test tube and mix
thoroughly. Observe any formation of precipitate. Test the solubility of the
precipitate in water.

Repeat the test using 70% alcohol instead of 95% alcohol. Compare the results.

H. Heat
SAFETY PRECAUTION:
While using water bath, use DISTILLED WATER instead of tap water. DO NOT
add too much water, so that the water will not overflow when boiling. Hot
water splash injuries can be minimized by wearing goggles and lab coats.

Put a pinch of powdered egg albumin in each of 2 dry test tubes labeled No.1
and No.2. To test tube No.1 – add 2 mL distilled water then place both test
tubes in a boiling water bath for 10 minutes, with constant shaking. Remove the
test tubes, cool to room temperature, and then add 2 mL distilled water to test
tube No.2. Filter the solutions and test both filtrates with Biuret reagent.

Perform Biuret test on 1 mL of 1% albumin solution (REFER TO Part A: Biuret

Test). Compare the results.

PROPER DISPOSAL: Dispose all used reagents into the sink under
running water.

QUESTIONS

1. Why do all proteins give a positive result for Biuret test?

2. Why does nitric acid stain the skin with a yellow color?
3. Will methionine give a positive result for the sulfur test? Explain.
4. What grouping in amino acids or proteins is responsible for the ninhydrin
reaction?
5. What color will be produced by proline with ninhydrin? Why?

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 8
Laboratory Manual

Name :_______________________________ Subject/Section :___________

Course/Year:_____________ Date Performed :___________
ID Number:______________

Data Sheet
Proteins and Amino Acids: Activity

Color Reactions 5

Data

A. Biuret Test

Test Sample Reagent Added Observation

1% Albumin
5 drops 10% NaOH +
1% Glycine
3 drops 1% CuSO4
Urea (s) - control

B. Millon’s Test (SEARCH FOR THEORETICAL RESULTS)

Test Sample Reagent Added Observation

1% Albumin
6 drops Millon’s
1% Gelatin
reagent
1% Phenol - control

C. Xanthoproteic Test

Test Sample Reagent Added Observation

1% Albumin 5 drops conc. Nitric
acid +
1% Gelatin
3 drops NH4OH
1% Phenol - control

D. Test for Sulfur (SEARCH FOR THEORETICAL RESULTS)

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 9
Laboratory Manual

Test Sample Reagent Added Observation

1% Albumin 5 drops 10% NaOH +
2 drops 5%
Gelatin (s) Pb(CH3COO)2

E. Hopkins-Cole Test

Test Sample Reagent Added Observation

1% Albumin 10 drops Hopkins-
Cole reagent +
1% Gelatin 10 drops conc. H2SO4

F. Ninhydrin Test

Test Sample Reagent Added Observation

0.2% Albumin

Ammonia 10 drops (or 0.5mL)

0.1% Ninhydrin
0.2% Urea reagent
0.2% Glycine

G. Alcohol Test

Test Solutions Reagent Added Observation

1% Albumin
95% Ethyl alcohol
1% Gelatin

1% Albumin
70% Ethyl alcohol
1% Gelatin

H. Heat

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.
Biochemistry (BTBIMOLAB) 10
Laboratory Manual

Test Solutions Reagent Added Observation

+ Distilled H2O, Δ, filter
To filtrate
+ 10% NaOH
+ 1% CuSO4
Powdered Albumin
Δ, + Distilled H2O, filter
To filtrate
+ 10% NaOH
+ 1% CuSO4

Natural Sciences Department, College of Science Information Technology and Engineering, Ateneo de
Zamboanga University, Zamboanga City, Philippines.