Nursing Biochemistry (NurBio) 1

Laboratory Manual

Laboratory Procedure Activity

Proteins and Amino Acids: 7
Precipitation

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
+ 2+ 2+ 2+
Ag , Hg , Cd and Pb 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
NH + NH +
3 3

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
+ + -
NH3 picrate
NH3
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.
Nursing Biochemistry (NurBio) 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 (SEARCH FOR THEORETICAL RESULTS)

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.
Nursing Biochemistry (NurBio) 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

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 2 mL of concentrated nitric acid in a test tube. Incline the test tube and
carefully add 1 mL of 0.5% albumin solution. 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.
Nursing Biochemistry (NurBio) 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 (REFER TO ACTIVITY

6 PROCEDURE: Biuret Test). 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.
Nursing Biochemistry (NurBio) 5
Laboratory Manual

Name :_______________________________ Subject/Section :___________

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

Data Sheet Activity

Proteins and Amino Acids: 7
Precipitation

Data

A. Salts of Heavy Metals (SEARCH FOR THEORETICAL RESULTS)

Test Added Reagent(s) Observation
Solutions
A precipitate has formed and dissolves after
1% Pb(CH3COO)2 further addition of the reagent
A precipitate has formed and dissolves after
1% Albumin 1% HgCl2 further addition of the reagent
A precipitate has formed and dissolves after
1% Cd(NO3)2 further addition of the reagent

B. Alkaloidal Reagents
Test Added Reagent(s) Observation
Solutions

After the first drop no precipitate has formed but

Saturated Picric acid after the second and third drop, a precipitate has
formed.
It already formed a precipitate on the first drop
and formed even more precipitate after the
1% Albumin 10% Tannic acid second and third drop.
One drop was enough to form cloudiness, after the
10% Trichloroacetic second and third drop more precipitate was formed.
acid

C. Inorganic Acids
Test Added Reagent(s) Observation
Solutions
Faint Precipitate has formed after the first drop,
Conc. HCl more precipitate formed after three more drops.
 With just one drop, formation of faint precipitate
can be seen, after more drops, greater cloudiness
1% Albumin Conc. H2SO4 is seen.
Precipitate has formed upon first drop, after more
Conc. HNO3 drops, greater cloudiness can be seen.

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

D. Heller’s Test

Test Added Reagent(s) Observation

Solutions
Upon adding the 0.5% Albumin to nitric acid,
separation of layers can be seen, the upper
0.5% Albumin layer is cloudy.
It formed a separation of layer, cloudiness
0.25% HNO3 formed in the upper layer.
Albumin
Two solution were almost uniform, lesser
0.125% cloudiness can be seen.
Albumin

E. Alcohol

Test Added Reagent(s) Observation

Solutions
1% Albumin Cloudiness has formed.
95% Ethyl alcohol No change in appearance,
1% Gelatin
1% Peptone No change in appearance.

1% Albumin Turned into a cloudy solution

1% Gelatin 70% Ethyl alcohol No change in appearance
1% Peptone No change in appearance

F. Heat

Test Added Observation

Substance Reagent(s)/Treatment
The color changed into dark purple which
indicates a positive result for the presence of a
+ Distilled H2O, , filter peptide bond
Powdered To filtrate
Albumin + 10% NaOH
+ 1% CuSO4
The color changed into a lighter purple almost
the same as control but is still positive for
, + Distilled H2O, filter biuret test and contains a peptide bond.
Powdered To filtrate
Albumin + 10% NaOH
+ 1% CuSO4
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.