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CHM 112 Lab Procedure 7:

AMINO ACIDS AND PROTEINS


Objectives:
(a) to isolate a protein, casein, from milk
(b) to discriminate reactive functional groups of different proteins and amino acids from
chemical tests

Proteins are biomolecules involved with every aspect of life on the planet. They can act
as structural materials (like in hair, feathers, hoofs and claws), reaction regulators that
control the rate of biological reactions (enzymes), transport molecules in blood and cell
membranes, receptors for cells, blood coagulation factors and hormones (such as
insulin and growth hormone), as well as many other functions. Proteins are one of the
three classes of biomolecules (along with carbohydrates and lipids) that provide energy,
structural components, and regulatory functions for living organisms.

Proteins, like many other biomolecules, are polymers; proteins are made up of amino
acid monomers. Each amino acid contains two functional groups -- an amino group (-
NH2) and a carboxylic acid group (-COOH) -- attached to the same carbon. By the
way, the carbon atom that has both the amino group and the acid group attached to it
is called the “α-carbon”, so all amino acids with this structure are called “α-amino
acids”. Repeated reaction of the carboxylic acid group with the amino group of the
next amino acid, forming a peptide bond, creates the polymer. Because proteins are
polymers with amino acids linked together by peptide bonds, they are often referred to
as polypeptides.

general amino acid structure

amino group H O carboxylic acid


group
H2N C C
OH
R

the side chain the α−carbon

This “backbone” structure, consisting of the amino group, the α-carbon and the
carboxylic acid group, is present in all amino acids and is required for the
polymerization reaction which forms a the protein. The side chain is the variable part of
the amino acid and accounts for many of the different properties of any amino acid.

There are 20 biologically important amino acids – these comprise the alphabet from
which the protein “words” and “sentences” are created. While many more amino acids
can be identified, these additional amino acids are most often modifications of one of

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the original 20 amino acids. The specific sequence in which amino acids are put
together defines the structure (and therefore the function) of the protein molecule.

Almost all the 20 important amino acids (except glycine) are chiral molecules. This
means that there is a carbon atom in the molecule that has four different groups
attached to it. This is sometimes referred to as chirality or handedness. If a model
of a chiral molecule is built with the familiar balls-and-sticks, two forms of the same
molecule can be built that are non-superimposable mirror images of each other (like
your right hand is the mirror-image of your left hand). These two molecules are called
enantiomers (or optical isomers) of each other, and they only occur in those
molecules with a chiral carbon (chiral center). These enantiomers have almost the
exact same properties – same melting point, same solubility in water, and the same
density. But they differ in that they rotate plane-polarized light in different
directions, and they will also react differently with other chiral molecules.
Sometimes a pair of enantiomers will have different tastes or odors from each other,
because they interact differently with taste receptors.

One enantiomer of a pair will rotate plane-polarized light to the right, and the other one
will rotate plane-polarized light to the left. The one that rotates light to the right is
designated as the D-enantiomer; the one that rotates light to the left is the L-
enantiomer. All the proteins found in living organisms on this planet are are
composed of L-amino acids (along with glycine). There are a few naturally occurring
D-amino acids on the planet, but they are not used in proteins; they are used in
bacterial cell walls and antibiotics produced by microorganisms.

Of all the 20 amino acids, a few are considered to be essential amino acids. These
are ones that cannot be synthesized by the body, and must be obtained through the
diet. The nine essential amino acids are: histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan, and valine. Arginine is considered to
be an essential amino acid for growing children, but not for adults.

Look at the amino acid glycine:

glycine H O

H2N C C
OH
H

alpha carbon

The α-carbon is attached to: an –NH2 group, a –COOH group, and 2 H atoms. It only
has three different groups attached to it (the H atoms count as only one type of
group, even though there are two of them). Glycine is not chiral.

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Now look at the amino acid serine:

alpha carbon
H O

H2N C C
OH
H C H

serine OH

The α-carbon is attached to the same –NH2 group and the same –COOH group and a
hydrogen as glycine. But the fourth group is the side chain: serine has a –CH2OH
group. Therefore, the α-carbon of serine is attached to four different groups.
Therefore it is a chiral center (or a chiral carbon). Other than glycine, all the 20
amino acids contain at least one chiral center (the α-carbon in the backbone), and
there are two amino acids – threonine and isoleucine – that contain a second chiral
center. The second chiral center is in the side chain.

H O H O

H2N C C H2N C C
OH OH
H3C C H H3C C H
isoleucine CH 2 CH 3
threonine
OH
second chiral second chiral
center center

Since amino acids contain an amino group, they are able to act as bases (proton
acceptors). Since they also contain a carboxylic acid group they are also able to act as
acids (proton donors) and form carboxylate ions. When in aqueous solution, these
functional groups may ionize, depending upon the pH of the solution. These are acid-
base reactions like any other acid-base reactions, where one portion of the molecule
accepts a proton and another portion donates a proton. Realize, of course, that the
proton from a carboxylic acid portion doesn’t necessarily donate to the amino group on
the same amino acid. One carboxylic acid group donates a proton to the closest amino
group, and an amino group accepts a proton from the nearest carboxylic acid group,
and these groups do not have to be on the same molecule. This ability of amino acids
and proteins to accept and donate protons allow them to act as buffers in the cell.

In a neutral solution, the amino group accepts a proton, and the carboxylic acid
donates a proton, forming what is referred to as a zwitterion; this form is the way

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amino acids always occur when in solution. This is a dipolar ion, with a charge at each
end. This is what threonine looks like as a zwitterion:

carboxylate ion
H O
+
H3N C C
-
O
H3C C H

OH
threonine -- ZWITTERION form

The side chains of amino acids may also participate in this proton donating and
accepting; the ionization of the side chain depends upon the pH of the solution. Acidic
amino acids (such as aspartic acid and glutamic acid) have a carboxylic acid functional
group in the side chain, which is also able to donate a proton:

aspartic acid in its ZWITTERION form aspartic acid in its ionic form
H O H O
+ +
H3N C C H3N C C
- -
O O
CH2 CH2
+
C H donated C
HO O -
O O

Basic amino acids (like lysine, arginine and histidine) have an amine group in the side
chain and are able to accept a proton:

histidine in its ZWITTERION form histidine in its ionic form


H O H O
+ +
H3N C C H3N C C
-
O O-
CH2 CH2
H+ accepted

H H
N N

N +N
..
H

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Each amino acid (as well as each protein) has an identifying pH value where the overall
molecule (amino acid or protein) has no net charge. This is known as the isoelectric
point (abbreviated as pI). Realize that at this pH value the molecule is not
uncharged, but rather, the molecule has NO NET CHARGE – positive and negative
charges exactly balance. Amino acids (or proteins) below the pI will have a positive
charge; those above the pI will have a negative charge. Look at aspartic acid and its
overall charge as the pH value of the solution goes from low to high:

H O H O
+ H O + H O
H3N C C OH -
+ OH -
H3N C C - OH-
OH H+
H3N C C - O H2N C C -
CH2 O H+ CH2 H+
O
CH2 CH2
C O - C O
HO C O O - C O
HO O
aspartic acid at pH = 1 aspartic acid at pH = 3 aspartic acid at pH = 4 aspartic acid at pH = 9
(overall charge = +1) (overall charge = 0) (overall charge = -1) (overall charge = -2)
ISOELECTRIC POINT

From the table given below, it is clear that there is a wide range of isoelectric points for
amino acids as well as proteins. It is also noteworthy that a protein is least soluble at
its isoelectric point. A protein can be soluble in a solution, but if the pH value equals
the protein’s isoelectric point, the protein will precipitate, often in clumps. This
technique is often used to remove proteins from a solution. These precipitated proteins
can be easily removed from a solution by filtering or centrifugation.

ISOELECTRIC POINTS (pI) OF SELECTED AMINO ACIDS AND PROTEINS

Amino acid pI Protein pI


Glycine 5.97 Silk Fibroin 2.2
Phenylalanine 5.48 Casein 4.6
Proline 6.30 Serum albumin 4.9
Methionine 5.74 Insulin 5.3
Arginine 10.76 Hemoglobin 6.7
Aspartic Acid 2.77 Lysozyme 10.7

Amino acids can link together (polymerize) to form a protein. The amino and carboxylic
acid functional groups on the backbone of the amino acids form amide bonds (in a
protein, these are often called peptide bonds) to create the sequence of amino
acids that is called the primary (1°) structure of the protein. These amide bonds
are the defining factor in the primary structure of the protein.

This peptide bond is formed in the same manner as any amide bond between an amino
group and a carboxylic acid group. This is realistically an acid-base reaction to form a
“salt” (the peptide molecule) and water.

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one residue of a protein

H O H O H O
H H O
+ + H +
H3N C C H3N C C N C C
-
O N C C
- O-
R1 H O R1 H R2
R2
H2O
peptide (amide) bond

The carboxylate group on the second amino acid is now available to react with the
amino group of a third amino acid, continuing the polymerization to form the protein. A
single amino acid in a protein is called a residue.

It should be noted that, in living cells, proteins only polymerize in one direction – from
the free amino group to the free carboxylate group (as shown in the diagram). While
most synthetic polymers can grow in both directions, proteins can only polymerize in
one direction. This is due to the specialized organelle in the cell – the ribosome – that
builds proteins, but only in one direction.

Once the primary structure of the protein is built, other types of interactions take place
to give the protein its final form. A protein is not just a long noodle that wiggles around
like a worm. All useful proteins have several layers of structure that enable them to
effectively perform their function.

Secondary (2°) structure is the regular patterns of protein chain segments that are
shaped by hydrogen bonds between backbone atoms. Two types of 2° structures
are the α-helix (a clockwise coil) and the β-sheet (a pleated sheet). Look in your text
for examples of these 2° structure types.

Tertiary (3°) structure is the manner in which the protein chain folds into its three-
dimensional shape (the protein’s conformation) due to interactions between side
chain atoms. These interactions can bring together portions of the protein chain that
are far apart in the 1° sequence of the protein. Two types of tertiary structure are:
fibrous proteins and globular proteins. Fibrous proteins (fiber-like structures) are
tough and insoluble – forming hair, feathers and fingernails. The collagen that makes
up hides of animals, tendons and many connective tissues is an example of a fibrous
protein. Globular proteins (compact ball-like shapes) are soluble – forming enzymes,
hormones, and transport proteins. Most of the interactions that make up the 3°
structure of proteins are non-covalent – such as ionic interactions between positively
charged and negatively charged side chains, and hydrogen bonding between polar side
chains. There is one special type of tertiary structure interaction that is a covalent –

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the disulfide link or bridge. This is formed from the reduction of two thiol groups
on the side chains of cysteine amino acids.
one cysteine residue

H H O H H H O H
N C C N N C C N
H C H H C H
SH S
a disulfide bond
SH oxidation S
H C H H C H
N C C N N C C N
H O H H H O H H
a second cysteine residue

Quaternary (4°) structure is the type of protein structure that is formed when two or
more protein chains are assembled into a larger three-dimensional structure. They are
held together by non-covalent interaction, most often between side chains of the
various amino acids in the different chains. Many proteins (including hemoglobin, that
transports oxygen in blood) are made up of more than one chain. Each chain is called
a subunit of the whole protein structure.

A protein that has folded properly and is functional is called a native protein. A
protein that functions using only the amino acids in its sequence is called a simple
protein, but a protein that requires additional non-amino acid groups to aid in its
function is called a conjugated protein. Hemoglobin is an example of a conjugated
protein – it has the heme group (a complicated porphyrin organic ring system which
contains an iron atom to coordinate the carrying of oxygen) embedded in the four
protein chains that make up the protein. Conjugated proteins may utilize a metal ion,
or a carbohydrate molecule or other co-enzyme factor, as the additional non-amino
acid group that is needed for protein function.

A protein is able to function because of a very delicate balance of forces (e.g. hydrogen
bonds) that maintain the protein’s structure. Since a protein’s 1° structure is based
upon covalent bonds, it is less likely to be disrupted, but the 2°, 3°, and 4° structures
are primarily based upon delicate, non-covalent bonds (the disulfide bridge being the
exception). If that balance is disturbed, the protein can lose its structure, and
therefore, its ability to function.

Disruption of the 2°, 3°, and 4° structures of a protein is called denaturation of a


protein, and often means that the protein’s structure and function are lost.

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Denaturation does not alter the protein’s 1° structure, but instead disrupts the forces
that contribute to the protein’s conformation. Denaturation can be irreversible or
reversible; sometimes an unfolded protein, under the proper conditions is able to
spontaneously return to its native state. Other times, the structure and function are
lost forever; an example of irreversible denaturation is the cooking of an egg. The
white of an egg contains the protein albumin, and once it is cooked, it cannot return
to its previous, native state.

Several things can cause protein denaturation.

1. Heat can denature proteins by increasing the energy of the atoms in the protein
so that the weak side-chain and backbone hydrogen bonds are disrupted.
Cooking an egg is an example.
2. Mechanical agitation can also denature proteins; an example of this is again
the egg white. When whipped, the peaks of meringue that form are due to
denatured albumin in the egg white.
3. A change of pH value can decrease the solubility of proteins by disrupting the
ionic attractions that form in the proteins 3° structure. This is why a protein
precipitates at its pI (isoelectric point). The ionic attractions have been broken
and the conformation is lost.
4. Inorganic salts (like ammonium sulfate, (NH4)2SO4) and certain metal ions
can also disrupt the same ionic attractions and denature proteins.
5. Detergents and organic solvents denature proteins by disrupting the
association between the side chains.

If properties of a protein are to be studied, the protein must be isolated from its
sample. In this procedure, you will precipitate the nutrient storage protein, casein,
from a sample of milk and then study chemical characteristics of its primary structure.
By adjusting the pH value of the milk sample to casein’s isoelectric point, the protein
will denature and precipitate.

A) Precipitation of casein from milk

Casein is precipitated from milk by adjusting the pH of a sample of milk to casein’s


isoelectric point (pH = 4.6) by adding glacial acetic acid. The impure precipitated
casein is separated by filtering through cheesecloth followed by washing first with
95% ethanol followed by a wash with a solution of 50% ethanol/50% diethyl ether
to remove any lipids. The casein is then dried. This casein will then be used along
with several other protein and/or amino acid solutions for the tests described below.
Since the casein is now denatured, no studies for protein function will be performed.
Only studies based upon the primary structure will be studied.

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B) Tests for Amino Acids and Proteins

1. BIURET TEST
Principle: Copper (II) ions, in basic solution, will complex with the nitrogen
atoms of four peptide (amide) bonds to form violet colored complex products. A
general illustration of the reaction is:

C
O
C O
O C
NH C NH
C
C O
C
C Cu+2
C C
O C
C O
NH NH

C C C C
O
O

These peptide bonds do not have to be in any order or any sequence in the
protein. Any four peptide bonds can coordinate with the copper ion to form the
color. A positive reaction for the biuret test is the formation of the violet color
that indicates the presence of peptide bonds. Solutions without peptide
bonds (such as free amino acids) will not form the violet color and will test
negative in the biuret test for the presence of peptide bonds.

2. NINHYDRIN TEST
Principle: Molecules with a free –NH2 group (a free amino group) will form a
purple-blue color with the ninhydrin reagent. If all amino groups are part of a
peptide bond, no color will result.

O
H
OH H O
2 + N C C RCHO + CO2 + 3 H2O +

OH H R OH
O O
O
ninhydrin
N

O O
purple-blue complex

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A positive ninhydrin reaction is the purple-blue color and indicates the
presence of free amino groups. Only FREE amino groups (like those in free
amino acids and protein side chains) will give a positive reaction with the
ninhydrin reagent. An exception is the amino acid proline, which contains a
secondary amino group (instead of the usual primary amino group found in
most amino acids and amino acid side chains) and will produce an orange color
instead of the purple-blue.

Solutions with no (or very few) free amino groups will not produce the purple-
blue color, and hence, the ninhydrin test is interpreted as being negative for the
presence of free amino groups. Protein chains have a free amino group at the
beginning of the protein, but this may not be enough to produce a positive
reaction.

3. XANTHROPROTEIC TEST:
Principle: Concentrated nitric acid (HNO3) will react with phenyl groups
(aromatic rings) to produce a yellow color. The amino acids tyrosine,
tryptophan and phenylalanine and any proteins that have significant
numbers of tyrosine and phenylalanine in their primary sequence will generate a
yellow color. Nitric acid will turn your skin yellow, and this reaction with phenyl
groups in your skin is responsible for the yellow color. This is not a toxic
reaction, but the yellow color doesn’t disappear until the skin cells are replaced.

H + H O H + H O

H N C C H N C C
+ HNO3
H O- H O-

nitrated phenyl group


has yellow color
NO2
phenylalanine

4. HEAVY METAL IONS TEST:


Heavy metal ions can react with proteins and some amino acids, causing them to
precipitate (heavy metals can denature proteins by disrupting the 2°, 3° and 4°
structures of proteins). The heavy metals capable of doing this are: Ag+, Cd+2,
Cu+2, Fe+3, Hg+2, Pb+2, Sb+3, and Zn+2.

This property of proteins is utilized when someone accidentally swallows a solution


containing one of these heavy metals; one of the antidotes is to give the person a
high-protein compound, often milk or raw eggs (if you look on the container of

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certain household products, you will see the directions “in case of accidental
swallowing, give milk or raw egg.”). The proteins in the milk/raw egg will bind with
the heavy metal and prevent absorption of the heavy metals in the intestinal tract.
This antidote doesn’t work with denatured, cooked egg; the native (non-denatured)
proteins are the species that react with and precipitate the heavy metals. The
following diagram is an example of an amino acid (either free or in a peptide chain)
reacting with the heavy metal, mercury.

O
H O H
C O N
CHR
+ + Hg2+
2 H3N C C Hg
O- 2+
O C
NH
R O

insoluble precipitate

Procedure:

Work in pairs in this entire procedure unless your instructor directs you to do
otherwise.

Part A: Isolation of the Protein Casein from Milk

1. Set up a warm water bath with a 600 mL beaker and approximately 200 mL of
tap water. Heat the water bath to about 40 °C, and maintain that temperature
(measuring with a thermometer).
2. Weigh an empty 250 mL Erlenmeyer flask (1) (do NOT use a side-arm flask for
this; use the 250-Erlenmeyer flask in you equipment locker) and add
approximately 50 mL of milk. Weigh the flask again (2) and determine the
mass of the milk (3).
3. Place the flask into the warm water bath for five minutes to heat the milk to 40
°C. Keep the flask in the water bath and, with constant stirring (using a glass
stirring rod and NOT the thermometer!), add about 15 drops of glacial acetic
acid (ethanoic acid). Large clumps of white precipitate should form (it should
look like curdled milk). If no precipitate forms, continue heating and stirring for
2 minutes. If precipitate still has not formed, contact your instructor. This
precipitate is denatured casein protein. See the illustration for the proper set up
for performing the precipitation.

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Apparatus for casein precipitation

4. When precipitation is complete, remove the flask (containing the precipitate)


from the water bath. Place a piece of cheesecloth over the opening of the flask
and use a rubber band to hold the cloth onto the flask neck (the cloth acts as a
filter). Invert the covered flask and drain as much liquid as possible from the
flask into a 150 mL waste beaker. You may need to turn the flask right side up
periodically to loosen precipitate that has stuck to the cloth. You may not be
able to drain all the fluid from the flask, but the more fluid removed, the less
lipid contamination will remain in the precipitate.
5. Remove the cheesecloth and add 25 mL of 95% ethanol to the flask
containing the precipitate. This is a washing step to remove more lipid
contamination from the precipitate. Stir the mixture for five minutes, and then
allow the precipitate to settle (about 3-4 minutes). Place the cheesecloth over
the neck of the flask (the same piece that was used in Step 4 may be used
again), and carefully decant the liquid into the same 150 mL waste beaker.
6. Remove the cheesecloth and add 25 mL of ether/ethanol mixture to the
flask containing the precipitate. Stir the mixture for 5 minutes; the solid
precipitate will be collected by vacuum filtration.
a. Set up the vacuum filtration (using the side-arm flask) apparatus as
shown in the illustration. Be sure to use the ring and ring stand so that
the apparatus is stabilized and won’t tip over. Be sure to put filter
paper into the funnel or all the precipitate will go through the
funnel and the filtration will have to be repeated.
b. Before hooking up the side-arm flask to the heavy vacuum tubing, turn on
the water and check that the water flow creates suction in the tube. If
not, try the tubing at another sink.
c. After the suction is verified, hook up the side-arm flask to the heavy
vacuum tubing, and turn on the water. The flow of the water into the

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sink will set up a vacuum and pull the filtrate through the filter and into
the flask. You may need to turn the water on full force to obtain the
proper suction. You may also need to press the filter into the flask to
generate the best seal and provide the proper suction.

Apparatus for vacuum filtration

d. Draw as much liquid through the filter as possible to dry the precipitate.
You may need to gently stir the precipitate (with a glass stirring rod) in
the filter to release liquid, but do this very gently. Otherwise the filter
paper becomes torn and precipitate will be lost into the filtrate liquid.
e. Spread the almost-dry precipitate in the filter onto a paper towel and let it
dry completely (about 10 minutes). Scrape the dry casein onto a tared
weighing paper and determine the mass of the dried casein (4). If, while
weighing the casein, the balance reading continues to drop and does not
stabilize, it means that some of the volatile ether/ethanol mixture is
continuing to evaporate; the sample is not yet completely dry. Let the
casein dry for five more minutes on a paper towel and again determine its
mass.
7. Determine the percentage of casein in the milk sample (5):

% casein = mass of dried casein x 100%


mass of milk sample

8. Save the dried casein for the chemical tests in Part B. Discard the filtrate in the
side-arm flask and the solution in the waste beaker into the appropriate waste
container (DO NOT DISCARD INTO THE SINK!).

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Part B: Chemical Properties of Proteins
**You need look up the composition of each of the five test substances to
determine if they are proteins polymers or free amino acids.

**Mix the bottle of casein solution before using for best results

**When mixing the dried casein and water for each test, break up the pieces
of casein with a stirring rod for best results.

1. Biuret test: Place 15 drops of each of the following solutions into five clean,
labeled test tubes:

a. 2% glycine
b. 2% gelatin
c. 2% albumin
d. dried casein from part A: about 0.2 grams + 15 drops deionized water (break
up pieces with stirring rod for best results)
e. 1% tyrosine

To each of these test tubes, add 5 drops of 10% NaOH and mix. Add two drops of 5%
CuSO4 to each tube and mix. The development of a purplish violet color is evidence of
the presence of peptide bonds. A deepening of the blue color (without a change to
purplish violet) is a negative test result. Record observations and results in your
notebook.

2. Ninhydrin test: Place 15 drops of each of the following solutions into five clean,
labeled test tubes:

a. 2% glycine
b. 2% gelatin
c. 2% albumin
d. dried casein from part A: about 0.2 grams + 15 drops deionized water (break
up pieces with stirring rod for best results)
e. 1% tyrosine

To each of these test tubes, add 5 drops of ninhydrin reagent and mix. Heat the tubes
in a boiling water bath for 5 minutes. The development of a purplish violet color is
evidence of the presence of free –NH2 groups (free amino groups; amino groups not
involved in peptide bond formation). Record your observations and results in your
notebook.

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3. Xanthroproteic test: Perform this test under a hood or close to the ventilation
grating! (This test uses concentrated nitric acid, which can cause burns if gotten on
the skin. Be careful with this reagent) Place 15 drops of each of the following
solutions into five clean, labeled test tubes:

a. 2% glycine
b. 2% gelatin
c. 2% albumin
d. dried casein from part A: about 0.2 grams + 15 drops deionized water (break
up pieces with stirring rod for best results)
e. 1% tyrosine

To each test tube, carefully add ten drops of concentrated HNO3 while gently swirling.
Heat the tubes carefully in a warm (approximately 60 ºC) water bath. The
development of a yellow color indicates a positive test (some may turn such an intense
yellow that the color may appear dark green). Observe any color change (formation of
yellow color) and record your observations in your notebook. Some results may be a
more intense yellow color than other results. It is acceptable to report a “slight yellow”
or “intense yellow” as observations.

4. Heavy metal ion test: Place 2 mL (about 40 drops) of milk (not dried casein
protein) in each of three clean, labeled test tubes. Add five drops of each of the
following solutions to the corresponding test tubes as indicated here and mix:

a. Pb2+ [found as Pb(NO3)2]


b. Hg2+ [found as Hg(NO3)2]
c. Ca2+ [found as Ca(NO3)2]
d. Fe2+ [found as Fe(NO3)2]
e. Na+ [found as NaNO3]

The presence of precipitate (looking like curdled milk; look for flakes or precipitate in
the milk or on the sides of the test tube) indicates the presence of precipitation of
proteins by the particular metal cations. If the milk retains its original appearance, no
precipitation has occurred, and the metal ion has not denatured the protein. Record
your observations in your notebook.

Place the waste from each test in to the appropriate waste container. Any leftover
dried casein may be placed in the trash; it is not toxic.

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References:

1. Introduction to General, Organic and Biochemistry (seventh edition), M.


Hein, L. R. Best, S. Pattison, S. Arena. Published by Brooks/Cole, Thomson
Learning, Inc., 2001.

2. Molecular Biology of the Cell (third edition), B. Alberts, D. Bray, J. Lewis, M.


Raff, K. Roberts, & J. D. Watson. Published by Garland Publishing, Inc., New York
and London, 1994.

3. Handbook of Cellular Chemistry (fourth edition), A. Cohen. Published by


Kendall/Hunt Publishing, Dubuque, Iowa, 1995.

4. Fundamentals of General, Organic and Biological Chemistry (third


edition), J. McMurry, M. E. Castellion. Published by Prentice Hall, Inc. Upper
Saddle River, New Jersey, 1999.

5. Essentials of Biological Chemistry, L. Buckberry & P. Teesdale. Published by


John Wiley and Sons, Ltd., West Sussex, England, 2001.

6. “Protein in Milk and Other Foods,” Retrieved for the Word Wide Web March 1, 2005
(http://lincoln.pps.k12.or.us/lscheffler/ProteinMilk.html).

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Lab partner(s) ________________________ Notebook points/initials __________

Date:______________ Name:___________________________

REPORT FOR LAB PROCEDURE 7:


AMINO ACIDS AND PROTEINS

Part A: Isolation of Casein from Milk

(1) Mass of empty flask ____________ g

(2) Mass of flask and milk ____________ g

(3) Mass of milk ____________ g

(4) Mass of dried casein _____________ g

(5) Percentage of casein in milk _____________ %

Part B: Chemical Properties of Proteins


Data:
1. Biuret test: To determine the presence of peptide bonds
Substance tested Observations Do observations
indicate presence or
absence of peptide
bonds?
2% glycine

2% gelatin

2% albumin

1% tyrosine

Dried casein solution

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2. Ninhydrin test: To determine the presence of free amino groups

Substance tested Observations Do observations


indicate presence or
absence of free
amino groups?
2% glycine

2% gelatin

2% albumin

1% tyrosine

Dried casein solution

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3. Xanthroproteic test: To determine the presence of phenyl groups

Substance tested Observations Do observations


indicate presence or
absence of phenyl
groups?
2% glycine

2% gelatin

2% albumin

1% tyrosine

Dried casein solution

4. Heavy metal ion test:

Metal ion tested Appearance of milk Did metal ion


denature the
protein?
Pb2+ [found as Pb(NO3)2]

Hg2+ [found as Hg(NO3)2]

Na+ [found as NaNO3]

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Answer the following questions about proteins and amino acids:

1. Fill in the blank questions:


a. The primary structure of proteins is determined by its ________________.

b. Disruption of the characteristic three-dimensional shape of a protein is


called _________________________.

c. Two examples of secondary protein structure are_____________ and


______________.

d. Amino acids in neutral solution exist as dipolar ions called ____________.

2. The isoelectric point (pI) for the amino acid lysine is 9.74. What would be the
charge on lysine (positive, negative or neutral) if it were in a buffer solution with
pH 9.00? There is no need to draw the structure of the amino acid.

3. Look up the amino acid structures of glycine, serine, and alanine, and draw the
structure of a tripeptide where glycine is first, serine is second, and alanine is
third. Draw the structure in the proper zwitterionic form.

4. What do disulfide bonds do for a protein?

5. Look up and list two hormones that are protein molecules (other than insulin and
growth hormone). Hormones that are amino acid derivatives are acceptable
answers.

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