Biochemistry for Allied Medical Courses 1

Laboratory Manual

Laboratory Procedure
Proteins and Amino Acids: Activity

Precipitation 6

INTRODUCTION

Proteins are separated from solution by the process of precipitation. Precipitation is

brought about by a great number of chemical reagents such as: salts of heavy metals,
alkaloidal reagents, alcohol, acetone, inorganic acids, and saturated solution of
ammonium sulfate. Precipitation of proteins is also a normal function of the body. An
example is the change of fibrinogen to fibrin in blood clotting and the precipitation of
casein as curd during digestion of milk.

Since amino acids in proteins exist in the Zwitterion form, they will ionize as negative
species above their isoelectric point. The positive ions of heavy metal salts, such as Ag +,
Hg2+, Cd2+ and Pb2+ combines with the protein anion to form an insoluble metal
proteinate. The precipitation with heavy metal salts may be illustrated as:
H H
- + - -
R COO + Ag NO3 R COO Ag
+
+ HNO3
+ +
NH3 NH3

At pH below their isoelectric point, the proteins exist as positive species. With alkaloidal
reagents such as tannic acid, picric acid and tungstic acid, the proteins are precipitated
from solution as follows:
H H
- + - -
R COO + H picrate R
+
COOH
-
+ NH picrate
NH 3
3

Proteins are very sensitive to pH changes. Addition of an excess inorganic acid such as:
HNO3, HCl, H2SO4 disrupts the salt bridges which stabilize the tertiary structure of
proteins. When a molecule acquires a large positive charge, the ionizable groups present
repel each other and reduces the availability of the protein molecule to interact with the
solvent molecule.

Ethanol coagulates proteins. The addition of ethanol or acetone reduces the dielectric
constant or ionic strength of the aqueous solution. The water molecules which form a
protective coating around the protein molecule hydrogen-bonds with the alcohol
molecules resulting in an increased electrostatic attraction between protein molecules.
The protein molecules then coalesce.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Biochemistry for Allied Medical Courses 2
Laboratory Manual

A factor which affects proteins at varying degrees is heat. Heating an aqueous solution
of protein may cause precipitation. Precipitation here is due to the disorganization of the
protein molecules. The hydrogen bonds, ionic bonds and hydrophobic bonds are
destroyed because of increased molecular vibration and the protein undergoes
intramolecular rearrangement, rendering it insoluble, but more readily digestible. Such is
called denatured protein.

APPARATUS/MATERIALS CHEMICALS/REAGENTS

Borrow: Request:
Bunsen burner 26 mL concentrated nitric acid, HNO 3
Iron ring 15 mL 95% ethyl alcohol
Iron stand 15 mL 1% albumin
3 mL 1% cupric sulfate, CuSO4
7 mL 10% sodium hydroxide, NaOH
6 mL 1% neutral albumin
15 mL 70% alcohol
3 mL 1% lead (II) acetate, Pb(CH3COO)2
3 mL 1% mercury (II) chloride HgCl2
3 mL 1% cadmium nitrate, Cd(NO3)2
3 mL 1% filtered albumin
3 mL saturated picric acid
2 mL 10% trichloroacetic acid
2 mL concentrated hydrochloric acid, HCl
2 mL concentrated sulfuric acid, H2SO4
1 mL 1% gelatin
1 mL 1% peptone
1 mL 10% tannic acid
0.5 g albumin

PROCEDURE

A. Salts of Heavy Metals

Place 2 mL of neutral 1% albumin solution in each of 3 test tubes. Add the

following reagents to the test tubes drop by drop.
test tube 1 – 1% lead (II) acetate
test tube 2 – 1% mercury (II) chloride
test tube 3 – 1% cadmium nitrate

Shake the test tube after each drop is added. Count the number of drops needed
to produce a precipitate. Then add an excess of the reagent. Note whether the
amount of precipitate is increased or dissolved by an excess reagent.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Biochemistry for Allied Medical Courses 3
Laboratory Manual

B. Alkaloidal Reagents

Prepare 4 test tubes containing 2 mL each of 1% albumin solution. Add the

following reagents to the corresponding test tube using a drop at a time and
shaking after every addition.
test tube 1 – saturated picric acid solution
test tube 2 – 10% tannic acid solution
test tube 3 – 10% trichloroacetic acid solution
test tube 4 – 20% tungstic acid solution

C. Concentrated Inorganic Acids

Prepare 3 test tubes containing 1 mL each of 1% filtered albumin solution. Add

the following reagents, drop-wise, shaking carefully after each addition.

test tube 1 – concentrated hydrochloric acid

test tube 2 – concentrated sulfuric acid
test tube 3 – concentrated nitric acid

Note whether a precipitate is formed. Then add an excess of the reagent.

Observe the effect of the excess reagent on the precipitate.

D. Heller’s Test

Place 1 mL of 0.5% albumin solution in a test tube. Incline the test tube and add
carefully 4 mL concentrated nitric acid. DO NOT SHAKE. Note the white
precipitate at the junction of the two liquids.

Test the sensitivity of this reaction by repeating the test on different dilutions of
the 0.5% albumin with distilled water. Note the dilution ratio at which the
solution gives only a faintly perceptible reaction.

E. Alcohol

Place 1 mL of the following test solutions in separate test tubes: 1% albumin,

1% gelatin, and 1% peptone. Add 5 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 on 1% albumin solution using 70% alcohol instead of 95%
alcohol. Compare the results.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Biochemistry for Allied Medical Courses 4
Laboratory Manual

F. Heat

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. 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. What is denaturation?
2. Why is egg white used as an antidote for lead or mercury poisoning?
3. Explain the use of tannic acid and picric acid in the treatment of burns.
4. Can heat coagulation test be used to detect the presence of albumin in urine?
Explain.
5. Which is more effective as a disinfectant – 95% alcohol or 70% alcohol? Why?

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Biochemistry for Allied Medical Courses 5
Laboratory Manual

Group No.___ Date Performed :___________

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

Data Sheet
Proteins and Amino Acids: Activity

Precipitation 6

Data

Name of Test Test Reagents No. of Effect of Excess

Solutions Added Drops Reagent
Added to
Produce
Precipitate

A. Salts of 1% Albumin 1%
Heavy Metals Pb(CH3COO)2

1% HgCl2

1% Cd(NO3)2

B. Alkaloidal 1% Albumin Picric acid

Reagents

10% Tannic
acid

10% TCA

20%
Tungstic acid

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Biochemistry for Allied Medical Courses 6
Laboratory Manual

Name of Test Test Reagents Result Explanation

Solutions Added
C. Inorganic 1% Filtered conc. HCl
Acids Albumin

conc. H2SO4

conc. HNO3

D. Heller’s O.5% conc. HNO3

Test Albumin

0.25%
Albumin

0.12%
Albumin

Name of Test Test Reagents Result Solubility of

Solutions Added Precipitate in Water
E. Alcohol 1% Albumin 95% alcohol

1% Gelatin

1% Peptone

1% Albumin 70% alcohol

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Biochemistry for Allied Medical Courses 7
Laboratory Manual

Name of Test Test Reagents Result with Explanation

System Used Biuret Test
in
F. Heat test tube 1 Powdered
(mixed, Egg Albumin
heated, and
then Distilled
filtered) Water

test tube 2 Powdered

(heated, Egg Albumin
mixed, and
then Distilled
filtered) Water

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