Experiment

Amino Acids and Proteins 8

Proteins are about 50% of the dry weight of most cells, and are the most structurally
complex macromolecules known. Each type of protein has its own unique structure and function.
Protein is a macromolecule made from amino acids; some consist of one‚ long polypeptide chain
while others are made from several intertwined polypeptides. It came from Greek word proteios
meaning first place. To form protein, the amino acids are linked by dehydration synthesis to form
peptide bonds. The chain of amino acids is also known as a polypeptide. Some proteins contain
only one polypeptide chain while others, such as hemoglobin, contain several polypeptide chains
all twisted together. The sequence of amino acids in each polypeptide or protein is unique to that
protein, so each protein has its own, unique 3-D shape or native conformation. If even one amino
acid in the sequence is changed, that can potentially change the protein’s ability to function. For
example, sickle cell anemia is caused by a change in only one nucleotide in the DNA sequence
that causes just one amino acid in one of the hemoglobin polypeptide molecules to be different.
Because of this, the whole red blood cell ends up being deformed and unable to carry oxygen
properly. Proteins are polymers of about 20 amino acids (the monomer).

In the approximately-20 amino acids found in our bodies, what varies is the side chain.
Some side chains are hydrophilic while others are hydrophobic. Since these side chains stick out
from the backbone of the molecule, they help determine the properties of the protein made from
them. Most naturally-occurring amino acids are the l- form, whereas synthetically-produced
amino acids give a 50:50 mixture. Notice that these molecules are mirror images of each other,
thus there is no way you can rotate one molecule to make it look like the other.

Amino acids are the monomers that are linked through peptide bonds to form a polymer
that we call a polypeptide. Amino acids differ in their side chains, which we refer to as R groups,
and the R groups are attached to the alpha carbon of the backbone. Here's a really stripped down
structure of an amino acid that doesn't show any of the hydrogen atoms. The amino group on the
far left would usually be NH3 with a positive charge. The carbon marked with the letter alpha
would have a hydrogen atom and an R group bound to it, and the carboxyl carbon would be
typically a dissociated carboxylic acid (COO-).

Amino acids play central roles both as building blocks of proteins and as intermediates in
metabolism. The 20 amino acids that are found within proteins convey a vast array of chemical
versatility. The precise amino acid content, and the sequence of those amino acids, of a specific
protein, is determined by the sequence of the bases in the gene that encodes that protein. The
chemical properties of the amino acids of proteins determine the biological activity of the protein.
Proteins not only catalyze all (or most) of the reactions in living cells, they control virtually all
cellular process. In addition, proteins contain within their amino acid sequences the necessary
information to determine how that protein will fold into a three dimensional structure, and the
stability of the resulting structure. The field of protein folding and stability has been a critically
important area of research for years, and remains today one of the great unsolved mysteries. It is,
however, being actively investigated, and progress is being made every day.
Objectives:
1. To observe the different properties of proteins.
2. To identify the different amino acids using different tests.

Materials:
test tube Graduated cylinder
dropper 1L beaker
test tube rack aluminum foil
250 ml beaker 6 capillary tubes
Bunsen burner

Reagents:
1% albumin
1% gelatin
1% casein
1% glycine
95% ethanol
70% ethanol
40% ethanol
conc. HNO3
1% HgCl2
10% NaOH
1%CuSO4
NH4OH
1% gelatin
1% tyrosine
1% phenol
Millon’s reagent
1% tyrosine
1% tryptophan
Ninhydrin solution
conc. H2SO4
1% phenylalanine
1% methionine
 Procedure:

I. Protein Precipitation
A. Heat
In separate test tubes, place 1% albumin, 1% gelatin, 1% casein and !%
glycine. Heat these test tubes in a boiling water bath for 5 minutes. Record your
observations.

B. Alcohol

In separate test tubes, place 1ml each of 95% ethanol, 70% ethanol and
40% ethanol. In each test tube, add 1% albumin. Compare the degree of precipitation in
each test tube.

C. Concentrated Acid

In a test tube, place 1ml 1% albumin. Add 4ml conc. HNO 3. Do not
Shake! Observe for cloudiness at the junction of the two liquids.

D. Salts of Heavy Metals

In separate test tube, place 1% albumin. Add drop by drop of 1% HgCl 2.

Shake while adding. Record your observations.

II. Color Reactions

A. Biuret Test

In separate test tubes, place 10 drops of each of the following: 1%

albumin, 1% casein, 1% glycine. In each test tube, add 5 drops of NaOH and 3 drops of
1%CuSO4. Mix and record your observations

B. Millon’s Test

In separate test tubes, place 20 drops of each of the following: 1%

albumin, 1% gelatin, 1% tyrosine and 1% phenol. In each test tube, add 6 drops of
Millon’s reagent. Mix and place in a boiling water bath for 5 minutes. Record your
observations

C. Ninhydrin Test
 In separate test tubes, place 1ml of each of the following: 1% albumin,
1% tyrosine and 1% tryptophan. In each test tube, add 10 drops of Ninhydrin solution.
Mix and place in a boiling water bath for 5 minutes. Record your observations

D. Hopkin’s Cole Test

In separate test tubes, place 20 drops of each of the following: 1%

albumin, 1% gelatin, 1% tyrosine and 1% tryptophan. Incline the test tubes and carefully
overlay with 10 drops of conc. H2SO4. Note for the color at the junction. Record your
observations

E. Xanthoproteic Test

In separate test tubes, place 10 drops of each of the following: 1%

albumin, 1% gelatin, 1% tyrosine, 1% phenylalanine and 1% tryptophan. In each test
tube, add 5 drops of conc. HNO3. Mix and place in a boiling water bath for 5 minutes.
Record your observations.

F. Sulfhydril Test
In separate test tubes, place 10 drops of each of the following: 1%
albumin, 1% gelatin, 1% tyrosine and 1% methionine. In each test tube, add 5 drops of
10%NaOH and 2 drops of 5% PbAc.. Mix and place in a boiling water bath for 5
minutes. Record your observations.

III. Chromatographic Analysis of Amino Acids

A. Preparation of Developing Chamber

In a 1L-beaker, mix 20ml of 95% ethanol, 2.5ml of NH 4OH and 2.5ml DH2O.
Cover the beaker with an aluminum foil. Stand for 15 minutes.

B. Preparation of Paper Chromatogram

Cut out 20cm x 13 cm Whatman filter paper. Handle the filter paper with minimal
fingerprint in it. With a pencil, draw a lengthwise line 10mm from the bottom of the filter
paper. Make a light mark point of equal intervals using a pencil. Using capillary tubes,
apply 3 drops of amino acid at one point. On the 5 remaining spots, apply different amino
acids using different capillary tubes. Staple the edge of the filter paper. Avoid
overlapping!
 space
staple wire

Slowly immerse the cylindrical paper in the beaker with the spots of amino acids
at the bottom of the beaker. Cover the beaker with aluminum foil. Stand until the solvent
is 1cm away from the upper edge.

Remove the paper from the beaker. Remove the staple wires. Mark the position of
the solvent using pencil. Spray the paper with 0.2% ninhydrin solution. Dry the paper in
the oven at 110oC. Take out the paper from the oven when blue colors are already visible.

Calculate the Rf value for each amino acids using the following formula:

Rf = Distance traveled by the spot (mm)

Distance traveled by the solvent (mm)

ANSWER SHEET
Name: _______________________________________________ Date: ____________
Course, Year and Section: _______________________________ Score:
Instructor: ___________________________________________
Class Schedule: _______________________________________
Group No. ______ Lab. Gown ________ Head Cap: ____

Experiment 8
Amino Acids and Proteins

I. Protein Precipitation

Precipitating Agent Test Sample Observation

Heat 1% albumin

1% gelatin

1% casein

1%glycine

95% ethanol 1% albumin

70% ethanol 1% albumin

40% ethanol !% albumin

Conc. HNO3 1% albumin

1% HgCl2 1% albumin

II. Color Reaction

A. Biuret Test
B. Millon’s Test

C. Ninhydrin Test

D. Hopkin’s Cole Test

E. Xanthoproteic Test

F. Sulfhydril Test
 III. Chromatographic Analysis of Amino Acids

Amino acid Distance traveled Distance traveled

by the spot (mm) by the solvent Rf value
(mm)

Computation:

Attach paper chromatogram here:

Summary:
Name of test Principle Positive result Significance

Biuret test

Millon’s test

Ninhydrin test

Hopkin’s Cole
test

Xanthoproteic
test

Sulfhydril test

Questions for Research:

1. Why do all proteins give positive result to Biuret test?
2. Why do you think nitric acid stain human skin?

3. What do you mean by protein denaturation?

4. Which is more effective disinfectant, 95% or 70% alcohol?

5. Do all amino acids give positive result to Ninhydrin test? If not, give the
amino acid that will give negative result to this test.

Reference Materials: