Exercise 4 Coagulation and Denaturation of Protein: Objective

EXERCISE 4
Coagulation and Denaturation of Protein
Protein denaturation is commonly defined as any non-covalent change in the structure

of a protein. This change may alter the secondary, tertiary or quaternary structure of the
molecules. When using this definition it should be noted that what constitutes denaturation is
largely dependent upon the method utilized to observe the protein molecule. Some methods
can detect changes in structure while others require large alterations in structure before
changes are observed.
Proteins are precipitated from solutions by salts of heavy metals such as ferric chloride,
lead acetate, copper sulfate, etc., by certain acids like picric acid, tannic acid, phosphotungstic
acid; the so called alkaloidal reagents by concentrated solutions of such salts as ammonium
sulfate, sodium sulfate and sodium chloride and by solvents like ethyl and methyl alcohol.
Objective
-To determine which reagents are capable of precipitating protein
-To determine the reason for precipitation of proteins with such reagents
Materials
Test tube Test tube rack Pipette
Protein samples Alkaloidal reagents Inorganic acids
Alcohols Heavy metals
Procedure
I. A. Precipitation of protein by alkaloidal reagents
Principle: Organic acids like trichloroacetic, phophomolybdic, picric and tannic acid are
frequently used as precipitants. These compounds carry a large negative charge which
neutralizes a positively charged protein to form insoluble salt.
1. prepare 5 ml of test solution (albumin, gelatin, casein)
2. add 5 ml picric acid solution by drops
3. observe the changes produced
4. repeat procedure using trichloroacetic, phosphomolybdic and tannic acid
5. record the result as following
(-) clear (+++) heavy precipitate

(+) faint turbidity (++++) solid precipitate
(++) heavy turbidity
RESULTS
Test solution Turbidity
Casein
Albumin
Gelatin
Water
B. Precipitation of protein by inorganic acid

Inorganic acids also precipitate protein, Hence, nitric acid is used for detecting the
presence of protein in urine (Heller’s test)
1. prepare 5 ml of test tube with solutions
2. add concentrated HNO3 by drops until precipitation is completed
3. repeat procedure using the concentrated HCl, concentrated H2SO4
4. Observe the results immediately and 30 minutes thereafter.
RESULTS
Casein
Albumin
Gelatin
Water
C. Precipitation of alcohol
Principle: Alcohol precipitate proteins when the proteins are electrically neutral (pH
4.7). This accounts for the antiseptic property of alcohol. When allowed to stand in alcohol,
protein undergoes irreversible change and is coagulated. This is used in fixing tissues for
histological examination.
1. prepare 5 ml of the test solutions
2. add equal volume of 70% alcohol
3. repeat procedure with 95% alcohol, dilute HCl, and 10 NaOH
4. compare the results with the control (tubes with rest solution only)
RESULTS
Casein
Albumin
Gelatin
Water
D. Precipitation by heavy metals
Principle: Proteins are precipitated from an alkaline solution by salts of heavy metals like
mercury, silver and lead. The carboxyl radical in the protein molecules form insoluble salts with
alkaline. Such metallic salts are used as antiseptic and germicide by precipitating bacterial
protein.
1. prepare 5 ml of test solutions
2. add an equal amount of mercuric chloride
3. repeat the procedure using lead acetate and silver nitrate
RESULTS
Casein
Albumin
Gelatin
Water
II. Denaturation and coagulation of protein
Solutions of proteins when heated between 38 and 60 degrees centigrade undergo

slight intramolecular rearrangement giving rise to changes in chemical, physical and biologic
properties and become denatured.
Denatured proteins become readily digestible by proteolytic enzymes; it loses

antigenicity, enzymatic or hormonal activity.
When proteins are denatured they become insoluble at isoelectric points, causing them
to precipitate. The process called Flocculation or clumping together of the dispersed chains of
denatured protein. If a flocculated protein is heated further, the clumped chains become
matted together in a mass which is insoluble, not only at isoelectric but also over the entire pH
range. The process is called Coagulation.
1. place 1 egg on boiling water for 5 minutes

2. observe what happens to the egg
Guide Questions
1. What is the principle behind the use of AgNO 3 for cauterization of wounds?
2. What is the principle behind the use of egg white and milk as antidote of metallic poisoning?
3. What is the main difference between coagulation and denaturation?
4. What is the significance of protein coagulation in sterilization?
5. Why is 70% alcohol commonly used as antiseptic rather than 95% alcohol?
6. Give 5 types of protein and identify where they are found and their function.

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EXERCISE 4

Coagulation and Denaturation of Protein

Protein denaturation is commonly defined as any non-covalent change in the structure

(-) clear (+++) heavy precipitate

Test solution Turbidity

B. Precipitation of protein by inorganic acid

Test solution Turbidity

Test solution Turbidity

Test solution Turbidity

II. Denaturation and coagulation of protein

Solutions of proteins when heated between 38 and 60 degrees centigrade undergo

Denatured proteins become readily digestible by proteolytic enzymes; it loses

1. place 1 egg on boiling water for 5 minutes

4. What is the significance of protein coagulation in sterilization?

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