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PROTEINS
I. RATIONALE
Enzymes are proteins. Muscles are proteins. The tissue which holds you together is
made up of proteins. Proteins are one of the most important types of compounds in
your body and in all living things. In this exercise we want to look at some of the
properties and methods of identification of proteins.

II. PREREQUISITES
1. As required In GENERAL INFORMATION - LAB, be ready to turn in a COPY of your:

A. Equipment list

B. Flow Chart

2. Be ready to answer the following questions:

A. What functional groups occur at the ends of protein molecules?

B. Which part of the egg contains protein which you will want to save for this
exercise?

C. What does it mean to "coagulate"?

D. Chemically, what does "buffer" mean?

E. Sketch and describe the use of the equipment and apparatus in your desk to
be used for this exercise.

III. OBJECTIVES
When you are finished with this exercise you should be able to:

1. Describe the test for nitrogen in proteins.

2. Describe the test for sulfur in proteins.

3. Describe the techniques of coagulating proteins.

4. Describe the buffering ability of proteins.

5. Describe the effect ions of heavy metals on proteins.

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6. Describe the Biuret Test.

7. Describe the Xanthoproteic Test.

8. Describe the Hopkins-Cole Test.

V. EXERCISE
1. INTRODUCTION

Proteins are polymers of amino acids: hundreds of amino acids may be joined together
to form a single protein molecule. The general structure of a protein molecule is:

R O +Q R O S, R O
* 5 * * 5 * * 5
NH2-C-C-O- *NH-C-C-O-*NH-C-C-OH
H .Q H S-x H

The "x" may be as much as several hundred or more. You can see that the protein
contains an amino functional group (left end) and a carboxylic acid functional group
(right end).

The R of each amino acid represents other functional groups. These may be other
amines, other carboxylic acids, alcohols, alkane, benzene-like, or sulfur containing
functional groups. Proteins therefor undergo the reactions characteristic of these
various functional groups.

2. PREPARATION OF ALBUMIN SOLUTION

Carefully separate the white from the yolk of an egg. Breaking the egg into a funnel
sometimes works to do this.

Place the white in a 100 ml beaker. Dispose of the yellow in the Organic Waste
container.

Add 50 ml of water and stir gently with a glass rod for several minutes.

Allow the mixture to settle. (Do Part 3 while waiting.)

Then decant the top (supernate) and save it for the rest of the experiment. Pour the
lower portion down the drain.

The supernate contains the protein, egg albumin, which you will use in most of the
exercise.
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3. TEST FOR NITROGEN

Mix well 0.5 gm of a solid protein (not the protein from Part 2) with 1 gm of soda lime in
a test tube.

Heat very strongly in the flame of a Bunsen burner.

Moisten the end a strip of Red Litmus Paper and test the vapors in the tube as they
come off.

What happens?

All proteins contain nitrogen in amine groups. When heated with soda lime, the amines
are converted to ammonia. How is this related to the results of the test with Red
Litmus?

Waste Disposal:

Carefully place the entire test tube and its contents into the "Inorganic" container.

4. TEST FOR SULFUR

Add 1 ml of concentrated NaOH solution to 2 ml of your albumin solution (from Part 2)

in a test tube.

Mix and boil CAREFULLY for 2 minutes. DON'T LET IT BOIL OUT OR BOIL DRY.

Cool and add Glacial Acetic Acid (from the reagent bottle with the molded on label)
DROP BY DROP - mixing between each drop - until the solution just turns a piece of
Blue Litmus Paper to pink.

Add a few drops of Lead Acetate solution. What happens?

Lead sulfide is a dark, insoluble solid. Not all proteins contain sulfur. The odor of rotten
eggs is due to hydrogen sulfide which forms as the egg decomposes. The greenish
gray layer on the surface of the yolk of a boiled egg is due to the reaction between iron
in the yolk and sulfur in the white of the egg.
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Waste Disposal:

Carefully place the entire test tube and its contents into the "Inorganic" container.

5. COAGULATION OF PROTEIN

Protein which is changed from its normal form is said to be "denatured". Such
denatured protein often clumps together until it forms an insoluble chunk - it coagulates.

A. Carefully boil 2 ml of your albumin solution in a test tube for several minutes. What
happens?

B. Place 3 ml of your albumin solution into a small test tube and CAREFULLY pour 2 ml
of concentrated nitric acid down the INCLINED SIDE of the test tube so that it flows
under your solution to form a layer at the bottom. What happens?

This test, known as Heller's Ring Test, is used to detect albumin in the urine - a test to
see if the kidney is malfunctioning.

Waste Disposal:

Pour the contents of the test tube into a Temporary Waste container for disposal by
the Instructor.

C. Mix 2 ml of your albumin solution and 5 ml of 95% ethyl alcohol in a small test tube.
What happens?

Based on this observation, why is alcohol used as a disinfectant?

What do you think happens to your insides if you drink a lot of alcohol?
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Waste Disposal:

Pour the contents of the test tube into the "Organic" container.

6. PROTEINS AS BUFFERS

Place 5 ml of water and 5 drops of Congo Red indicator in a test tube.

Place 5 ml your albumin solution and 5 drops of Congo Red indicator into a second test
tube.

Then add to each, DROP BY DROP, (count the drops, and mix after each drop) a
solution of 0.1 M Acetic Acid until the solution turns dark.

How many drops are required to turn the solution dark for:

water: _____

albumin: _____

A "buffer" is a substance that resists a change in pH. When Congo Red changes from
reddish to a dark color (blue?) it indicates a change in pH. Which was the better buffer,
water or albumin solution?

Waste Disposal:

Pour the contents of the test tube into the "Organic" container.

7 REACTION WITH IONS OF HEAVY METALS

Place 2 ml of your albumin solution into a test tube and add 1% Mercuric Chloride drop
by drop until something happens. What happens?

Other metallic ions, such as those of lead and silver, cause a similar reaction.

If many proteins must be soluble in order to carry out their functions, why do you think
that compounds of mercury, lead or silver - "heavy metal ions" - are poisons?
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Waste Disposal:

Pour the contents of the test tube into the “Inorganic" container.

8. BIURET TEST

Proteins in solution can be detected by means of a number of color-producing

reactions. One of these is the Biuret Test, called this because proteins give a color
similar to that of a compound called biuret.

To 2 ml of your albumin solution add and mix in 3 ml of 0.5% NaOH.

Then add and mix in 10 drops of 1% CuSO4. What is the color that results?

Waste Disposal:

Pour the contents of the test tube into the "Inorganic" container.

9. XANTHOPROTEIC TEST

Many proteins contain amino acids which have aromatic (benzene-like) functional
groups. These functional groups will react with nitric acid in the xanthoproteic reaction.

Carefully add 1 ml of concentrated nitric acid to 2 ml of your albumin solution in a test

tube.

Stir well with a glass stirring rod. What happens?

You may have noticed during previous labs that if you were not careful with nitric acid
you got yellow spots on your fingers. Why do you think that happened?

Waste Disposal:

Pour the contents of the test tube into a Temporary Waste container for disposal by
the Instructor
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10. HOPKINS-COLE TEST

Add 2 ml of Hopkins-Cole Reagent to 1 ml of your albumin solution and mix.

VERY SLOWLY AND CAREFULLY pour this down the side of an inclined test tube
containing 2 ml of concentrated sulfuric acid, so that it forms a layer on top of the acid.

Allow the test tube to stand without mixing. What happens after about 5 minutes?

Waste Disposal:

Pour the contents of the test tube into a Temporary Waste container for disposal by
the Instructor