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Activity 6 Laboratory Procedure
Activity 6 Laboratory Procedure
Laboratory Manual
Laboratory Procedure
Proteins and Amino Acids: Activity
Precipitation 6
INTRODUCTION
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
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
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
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.
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
Data Sheet
Proteins and Amino Acids: Activity
Precipitation 6
Data
A. Salts of 1% Albumin 1%
Heavy Metals Pb(CH3COO)2
1% HgCl2
1% Cd(NO3)2
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
conc. H2SO4
conc. HNO3
0.25%
Albumin
0.12%
Albumin
1% Gelatin
1% Peptone
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Biochemistry for Allied Medical Courses 7
Laboratory Manual
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.