Nursing Biochemistry Laboratory (NurBio Lab) 1

Laboratory Manual

Laboratory Procedure Activity

Proteins and Amino Acids: 6
Color Reactions

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.

alpha carbon

variable group R CH COOH caboxyl group

NH2 amino group

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 CH3, 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 and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 2
Laboratory Manual

H H O H H O H H O H H O

N N N N
R1 R2 R3 R4

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 quarternary 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 and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 3
Laboratory Manual

H H
O

O C H H C O
N N
R ++ R
Cu
O O
N N
R CH H H CH R

O
H H
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, Na2S. 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 NH3 liberated combines with one mole
of the reduced ninhydrin and one mole of the oxidized ninhydrin to form Ruhemann’s
purple:

O O O

OH
several steps
+R CHCOOH N
OH NH2

O O O

ninhydrin purple dye

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 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 30 mL 1% albumin
Iron ring 25 mL 1% casein
Iron stand 20 mL 1% gelatin
15 mL 1% phenol
Bring: 12 mL 10% sodium hydroxide, NaOH
Marbles 12 mL Hopkins-Cole reagent
8 mL Millon’s reagent
8 mL concentrated ammonium hydroxide,
NH4OH
8 mL concentrated sulfuric acid, H2SO4
5 mL 1% peptone
5 mL 1% glycine
4 mL 1% copper (II) sulfate, CuSO4
4 mL 5% lead (II) acetate, Pb(CH3COO)2
2 mL 0.1% ninhydrin solution
2 mL 0.2% albumin solution
1 mL ammonia water
1 mL 0.2% urea
1 mL 0.2% glycine
8 mL concentrated nitric acid, HNO3
0.5 g urea
0.5 g gelatin
0.5 g peptone

PROCEDURE

A. Biuret Reaction

1. Place 10 drops each of the following in separate test tubes: 1% albumin, 1%

casein, 1% peptone 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
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.

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

B. Millon’s Test

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.

CAUTION: Always wear gloves before handling corrosive substances

! like concentrated acids and bases!

C. Xanthoproteic Test

1. Into 3 separate test tubes place 10 drops of the following: 1% albumin, 1%

gelatin, and 1% casein. Add 5 drops of concentrated HNO3 to each test tube. Mix
thoroughly. 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
NH4OH. 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.

D. Test for Sulfur

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.

E. Hopkins-Cole Reaction

1. Mix 10 drops of Hopkins-Cole reagent to 20 drops of the following solutions in

separate test tubes: 1% albumin, 1% casein, 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.

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

F. Ninhydrin Reaction

1. To 1 mL of neutral 0.2% albumin solution add 0.5 mL 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.

PROPER DISPOSAL: Dispose of solutions in the proper waste bottles (as

acid or basic wastes, and organic or inorganic wastes).

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 and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 7
Laboratory Manual

Group No.___ Date Performed :___________

Subject/Section:__________ Instructor’s Initials :___________
Members:
1. _____________________
2. _____________________
3. _____________________

Data Sheet Activity

Proteins and Amino Acids: 6
Color Reactions

Data

Name of Test Test Reagents Result

Solutions Added
A. Biuret 1% Albumin + 10% NaOH
Reaction + 1% CuSO4

1% Casein

1% Glycine

1% Peptone

Urea (s)

B. Millon’s 1% Albumin + Millon’s

Test (Search Reagent
for [HgNO3 and
Theoretical 1% Casein Hg(NO3)2
Results Only) added with
conc. HNO3]
1% Gelatin

1% Phenol

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 101: General and Inorganic Chemistry 8
Laboratory Manual

Name of Test Test Reagents Result

Solutions Added
C. 1% Albumin + conc. HNO3
Xanthoproteic + conc.
Reaction NH4OH
1% Casein

1% Gelatin

1% Phenol

D. Test for 1% Albumin + 10% NaOH

Sulfur + 5%
Pb(CH3COO)2
Gelatin (s)

Peptone (s)

E. Hopkins- 1% albumin + conc.

Cole Reaction H2SO4

1% casein

1% gelatin

F. Ninhydrin 0.2% + 0.1%

Reaction Albumin ninhydrin
solution
Ammonia
water

0.2% Urea

02%
Glycine

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