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Laboratory Manual in BIOCHEMISTRY for Nursing and Psychology Students

EXPERIMENT NO. 8

AMINO ACIDS, PEPTIDES, AND PROTEINS

Amino Acids

Amino acids are the building blocks of proteins. In class you learned the structures of the 20 common amino acids
that make up proteins. All amino acids have the general structure shown below.

Peptides

Peptides are short chains of amino acids, each one connected to the next by an amide linkage called a peptide bond.

Below is the chemical reaction by which two amino acids become connected by an amide linkage (the circled O and
two H‟s are eliminated as a water molecule during the reaction):

A tripeptide (composed of three amino acids):

The artificial sweetener aspartame (brand name Nutrasweet) is an example of a modified peptide. The structure of
aspartame is shown below.
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Proteins

Proteins are the work horses of living cells. They act as microscopic cellular machines that function in much the
same manner as human-built machines. Protein molecules are long chains of amino acids connected to each other in
the same manner as in peptides. Very small proteins may be composed of 50 to 100 amino acids, while large
proteins may contain thousands of amino acids. In a normal functional protein, the long chain of amino acids is
“folded” into a 3- dimensional shape (imagine the long amino acid chain as a piece of string that has been crumpled
up into a 3-D ball/blob). Every protein has a unique 3-dimensional shape that is suited to its biological function in a
living organism. If the 3-dimensional shape “unfolds” for some reason, the protein will no longer be able to carry
out its biological function and will usually be destroyed by the cell. (Unfolding is like the crumpled up blob of
string being unwound again.) The unfolding of a protein structure is called denaturation.

Chemical Reactions of Amino Acid and Protein Functional Groups

Certain functional groups in amino acids and proteins can react to produce characteristically colored products. The
color intensity of the product formed by a particular group varies among proteins in proportion to the number of
reacting functional, or free, groups present and their accessibility to the reagent. In this part of the experiment, you
will use various color-producing re- agents (dyes) to qualitatively detect the presence of certain functional groups in
amino acids and proteins.

Biuret Test

The Biuret reagent contains copper ions which give it a blue color. The copper ions will interact with a compound
that contains two or more peptide bonds, resulting in the formation of a violet/purple-colored product. When a
compound does not have at least two peptide bonds, it will not react with the Biuret reagent, and no purple color
will appear (solution will remain a shade of blue due to the copper ions).

Positive Biuret test: violet/purple product forms

Negative Biuret test: no violet/purple product formed

Ninhydrin Test

The ninhydrin reagent will react specifically with a primary (1) amino functional group on a compound, resulting in
the formation of a violet/purple-colored product. When a compound does not have a primary amino group, it will
not react with the ninhydrin reagent, and no purple color will appear (solution will remain colorless).

Positive ninhydrin test: violet/purple product forms

Negative ninhydrin test: no violet/purple product formed

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Denaturation Test

Strong acid will often denature (unfold) proteins. Proteins that are properly folded into their normal 3-dimensional
shape tend to be soluble in aqueous solution. However, proteins that have been denatured tend to clump together
and come out of solution as a precipitate because they are no longer soluble when they are denatured and clumped
together. In the denaturation test, strong acid is used to test a solution for the presence of protein. When strong acid
is added, the formation of a white precipitate (composed of denatured protein molecules) is considered a positive
denaturation test and indicates the presence of protein in the solution. If no precipitate forms, the denaturation test is
negative, and indicates that no protein is present in the solution.

Xanthoproteic Test

Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can undergo
reactions that are characteristic of ben- zene and benzene derivatives. One such reaction is the nitration of a benzene
ring with nitric acid. The amino acids tyrosine and tryptophan contain activated benzene rings and readily undergo
nitration. The amino acid phenylalanine also contains a benzene ring, but the ring is not activated and therefore does
not readily undergo nitration.

This nitration reaction, when used to identify the presence of an activated benzene ring, is commonly known as the
xanthoproteic test, because the prod- uct is yellow. Xanthoproteic comes from the Greek word xanthos, which
means “yellow.” The intensity of the yellow color deepens when the reaction occurs in basic solution. The
xanthoproteic test for tyrosine is shown below:

This reaction is one of the reactions that occurs if you spill a concentrated solution of nitric acid onto your skin. The
proteins in skin contain tyrosine and tryptophan, which become nitrated and turn yellow.

Millon’s Test

Millon’s test is a test specific for tyrosine, the only amino acid containing a phenol group, a hydroxyl group
attached to a benzene ring. In Millon’s test, the phenol group of tyrosine is first nitrated by nitric acid in the test
solution. Then the nitrated tyrosine complexes mercury(I) and mercury(II) ions in the solution to form either a red
precipitate or a red solution, both positive results. Proteins that contain tyrosine will therefore yield a positive result.
However, some proteins con- taining tyrosine initially form a white precipitate that turns red when heated, while
others form a red solu- tion immediately. Both results are considered positive. Note that any compound with a
phenol group will yield a positive test, so you should be certain that the sam- ple that you are testing does not
contain any phenols other than those present in tyrosine.

Hopkins–Cole Test

The Hopkins–Cole test is specific for tryptophan, the only amino acid containing an indole group. The indole ring
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reacts with glyoxylic acid in the presence of a strong acid to form a violet cyclic product. The reaction is shown
below.

The Hopkins–Cole reagent only reacts with proteins containing tryptophan. The protein solution is hydrolyzed by
the concentrated sulfuric acid at the solution interface. Once the tryptophan is free, it reacts with the glyoxylic acid
to form the violet product.

Nitroprusside Test

The nitroprusside test is specific for cysteine, the only amino acid containing a sulfhydryl group (–SH). The group
reacts with nitroprusside in alkaline solution to yield a red complex, as shown below.

EXPERIMENT NO. 8
AMINO ACIDS, PEPTIDES, AND PROTEINS

Group No. Name Course, Year & Section Date Score

Pre-Laboratory

1. Draw the structures of alanine and glycine.

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2. Show the structure of the peptide that would result from a chemical reaction between glycine and alanine.

3. Indicate whether each of the following is an amino acid, a peptide, or a protein. B. Indicate whether each
will give a positive or negative Biuret test.
A. Amino Acid, Peptide, Protein? B. Biuret test? (+ or - )

aspartic acid (a.k.a. aspartate) Amino Acid Negative

phenylalanine Amino Acid Negative

Nutrasweet (aspartame) Peptide Negative

4. Examine the structure of aspartame (Nutrasweet) in the introduction, paying careful attention to the side
chains. Which two amino acids would be released if the peptide bond was, hydrolyzed‟ (broken by a
reaction with H2O)?
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5. Describe the hazards associated with the following procedures.

a. xanthoproteic test

• This test is used to determine the amount of soluble proteins in a solution and uses concentrated
Nitric acid.

• The test also requires the use of NaOH, ammonium hydroxide solution

• Some of the reagents used in this test are hazardous and should be handled with extreme care. For
example Sodium Hydroxide (NaOH) and ammonium hydroxide are both highly corrosive.

• Concentrated Nitric acid (HNO3) reacts explosively with combustible organic or readily
oxidizablematerials. Moreover long term exposure to vapors that arise from HNO3 can have
adverse impact on the health.

b. millons test

• Millon's test is performed to detect the presence of soluble proteins.

• Million's test involves the use of Millon's reagent (Hg/HNO3) that gives positive result with
proteins containing phenolic amino acid tyrosine.

• Millon's reagent is hazardous to the aquatic environment, and repeated exposure can also cause
specific target organ toxicity. HNO3 present in the reagent is corrosive and Millon's reagent is also
toxic by inhalation. Millon's reagent can cause severe skin burns and eye damage.

c. hopkins-cole

• Hopkins-cole test is performed to detect the presence of tryptophan in the proteins.

• Hopkins test involves the use of glyoxylic acid and concentrated sulfuric acid.

• Hopkins cole reagent is zazardous in case of skin contact (irritant), of eye contact (irritant), of
ingestion.

• It contains Corrosive liquid, toxic (sulfuric acid, mercuric sulfate, solution),

d. nitroprusside test

• Nitroprusside test is performed to detect the presence of free thiol groups of cysteine in proteins.

• Potential hazards associated with Nitroprusside are: it may cause eye irritation and skin irritation.

• Upon accidental ingestion, it may cause irritation of the digestive tract and its metabolism may
release cyanide which can adversely affect the health.

• Accidental inhalation of nitroprusside may cause respiratory tract irritation.

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6. Briefly explain the meaning of the following terms:

a. essential amino acids

- Amino acids, often referred to as the building blocks of proteins, are compounds that play many
critical roles in your body.

- They’re needed for vital processes like the building of proteins and synthesis of hormones and
neurotransmitters.

- Some may also be taken in supplement form for a natural way to boost athletic performance or
improve mood.

b. peptide bond

- A peptide bond is a chemical bond formed between two molecules when the carboxyl group of one
molecule reacts with the amino group of the other molecule, releasing a molecule of water (H2O).

c. The R-group of the amino acid lysine is –CH2–CH2–CH2–CH2–NH2

- Lysine is basically alanine with a propylamine substituent on theβcarbon. The ε-amino group has a
significantly higher pKa (about 10.5 in polypeptides) than does the α-amino group.

- The amino group is highly reactive and often participates in a reactions at the active centers of
enzymes. Proteins only have one α amino group, but numerous ε amino groups. However, the
higher pKa renders the lysyl side chains effectively less nucleophilic. Specific environmental
effects in enzyme active centers can lower the pKa of the lysyl side chain such that it becomes
reactive.

7. Draw the complete structure of lysine, including the sites of ionization, in:

a. strongly acidic solution

b. neutral solution
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c. strongly basic solution

8. Predict the results of the following tests on lysine.

a. solubility in acidic solution

The structure of lysine is shown below

From the above structure , we can see that there are 2 amino groups in Lysine. Amines are soluble
in acids.

In acidic solutions, both the amino groups would get protonated, making the substance soluble.

Therefore Lysine would be soluble in acidic solution.

b. ninhydrin

Ninhydrin is the reagent used for testing presence of alpha amino acids in the given peptide or prote
in. It's a strong oxidizing agent. When ninhydrin which is originaly yellow in color mixed with
substance containing amino acids, it turns deep blue in color. Lysine being an alpha amino acid
would give a positive. Ninhydrin test turning the solution blue in color

c. biuret

Biuret test is the test used for confirming the presence of peptide bonds. In a protein, many amino
acids are joined together by a special type of linkage, called peptide bond. Shown below is the
peptide bond between 2 amino acids.
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Biuret test confirms the presence of this special linkage. In order to have positive biuret test we
need. Lysine molecules attached by peptide linkage. Since Lysine is just an amino acid, it will not give a
positive biuret test.

d. nitroprusside

Nitroprusside is the reagent used for detecting the presence of Sulfur containing groups such as thiol. A
positive test turns the solution deep red in color. Since there is no sulfur in lysine, it will give negative
test for nitroprusside.

PRINCIPLES BEHIND THE TEST

Color reactions:

1. SAKAGUCHI TEST: A general test for amino acids. Arginine residues in protein react with - naphthol and
sodium hypobromite to form a colored chromogen

2. NINHYDRIN TEST: Reagent will react specifically with a primary (1) amino functional group on a
compound, resulting in the formation of a violet/purple-colored product.

3. MILLON’S TEST: A test specific for tyrosine, the only amino acid containing a phenol group, a hydroxyl
group attached to a benzene ring

4. NITROPRUSSIDE TEST: Specific for cysteine, the only amino acid containing a sulfhydryl group (–SH).
The group reacts with nitroprusside in alkaline solution to yield a red complex

5. XANTHOPROTEIC TEST: Proteins undergo nitration with hot, strong HNO 3, producing a yellow cole in an
acidic and orange color in an alkaline solution

6. HOPKIN’S COLE TEST: Specific for tryptophan, the only amino acid containing an indole group. The
indole ring reacts with glyoxylic acid in the presence of a strong acid to form a violet cyclic product.

PROCEDURES:

2. COLOR REACTIONS:
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Prepare the six samples (casein, starch, albumin, gelatin, peptone and phenol)

a. Sakaguchi Test
1. To 1ml of the protein sample, add 3 drops of 40% NaOH and 4 drops of - naphthol solution (in 1%
alcohol)
2. After 3 minutes, add 10 drops of bromine water and observe the color change. Record.

b. Ninhydrin Test
1. To 1ml of the protein sample, add 10 drops of ninhydrin (0.2% in acetone) and heat to boiling.
2. Observe the color formation

c. Sulfur Test
1. To 1ml of the protein solution, add an equal volume of 40% NaOH and boil for 3 minutes.
2. Cool and add 2-3 drops of Lead acetate with mixing. Observe the color develop.
NOTE: If the solution turns black, or brown color, do the Nitroprusside test. If the solution turns
yellow, do the xantroproteic and Hopkin’s cole test

d. Millons
1. To 1ml of the protein solution, add 1ml of H2SO4 solution (10% solution in 10% sulfuric acid)
2. Boil gently for 30 seconds and add 2ml of 1% sodium nitrate solution
3. Observe color changes

e. Nitroprusside
1. Place 1ml of protein sample to 0.5 ml nitroprusside
2. Add 2ml of diluted ammonium hydroxide solution and observe the color change/develop

f. Xanthoproteic
1. To 1ml of the protein sample, add dropwise 1ml of concentrated HNO 3
2. Heat to boiling and cool in tap H2O and observe the formation of a colored precipitate
3. Add 2 ml of 10% NaOH and note the color change

g. Hopkin’s Cole
1. To 1ml of the protein sample, add 1ml of glyoxylic acid (Hopkin’s Cole reagent) and swirl gently
2. Incline the test tube and carefully add dropwise 1ml of concentrated H 2SO4
3. Note the colored ring at the junction of the two layers of liquids

Protein Sample

 
color reaction thin layer chromatography

   
sakaguchi test ninhydrin test sulfur test millon’s test
 
   
brown yellow pink yellow
 
nitroprusside  
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Xanthoproteic Hopkins Cole

Schematic Diagram showing the test procedures for the identification of unknown amino acids

DATA AND RESULTS:

1. State the different classification schemes for amino acids

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2. What is the biological importance of amino acids?

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TABULATED RESULTS

UNKNOWN SAMPLES SAKAGUCHI NINHYDRIN MILLON’S SULFUR

1 (Casein)

2 (Starch)

3 (Albumin)

4 (Agar)

5 (Phenylalanine)

6 (Peptone)

7 (Tyrosine)

8 (Glycine)

UNKNOWN SAMPLES NITROPRUSSIDE XANTHOPROTEIC HOPKIN’S COLE

1 (Casein)
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2 (Starch)

3 (Albumin)

4 (Agar)

5 (Phenylalanine)

6 (Peptone)

7 (Tyrosine)

8 (Glycine)

EXPERIMENT NO. 9

ANALYSIS OF MILK

Milk is considered to be a complete food, since it contains almost all the nutrients needed for humans. It
contains carbohydrates, proteins, fats, many vitamins and minerals. However, it is deficient in iron, copper and
Vitamin D.

Milk is the secretion of lactating mammary gland12ch. Cow’s milk contains 4.0% proteins, 3.5%
triacyglycerols, 4.6% lactose and 0.75% minerals.

The major proteins in milk are casein, lactalbumin and lactoglobulin. Casein makes up about 80% of the
protein in cow’s milk and aabout 40% of that in human milk. The casein in milk is a phosphoprotein containing
about 0.7% phosphorus. It is present as calcium caseinate in cow’s milk and as potassium caseinate in human milk.
Minerals in milk include calcium (180mg% in cow’s milk), phosphate, sodium, potassium and chloride
ions. Whiteness of milk is due to emulsified fat. The pH of fresh milk is 6.6 – 6.8 and its specific gravity is 1.03

PROCEDURES

PRECIPATATION OF CASEIN
1. Dilute 10 ml of fresh milk with an equal volume of water. Add 5ml of 10.0% acetic acid drop by drop with
slow stirring. Casein precipitates along with the associated fat.
2. Filter the suspension. Perform the following tests with wet precipitate

TEST FOR FAT

a. To part of the precipitate, add 3.0ml ethanol and warm in a water bath for 2 minutes
b. Add 2ml of water and filter into a test tube Fat dissolves in ethanol precipitates

TEST FOR CASEIN

a. Suspend rest of the precipitate in 5.0ml water.
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b. Using 1ml of the substance, perform Biuret test using 1ml of Biuret reagent ( or add 1ml of 10% NaOH
and 4 drops of CuSO4 solution. Mix and note the color that develops)

TEST FOR LACTALBUMIN AND LACTOGLOBULIN

a. Perform Biuret test. Use 1ml of fresh milk.

PRECIPITATION TEST
a. Perform precipitation test with heat and acetic acid. Obtain 1 ml of fresh milk, hold the test tube over a
flame in slanting position and allow the upper part to reach boiling point. The lower part serves as the
control.
b. Add few drops of 10.0% acetic acid without mixing. Observe.

TEST FOR LACTOSE

a. Perform Benedict’s Test. Use 1ml of fresh milk and add 1ml of the benedict reagent.
b. Place the test tube in a boiling water for 5 minutes. Observe and record the results.

TEST FOR CALCIUM

a. To 1ml of fresh milk, add 15 drops of 10.0% acetic acid and 1 ml of 2% potassium oxalates solution.
Observe and record results.

TEST FOR INORGANIC PHOSPHATES

a. To 1ml of fresh milk, add a few drops of concentrated nitric acid in an inclined position
b. Add a pinch of ammonium molybdate.
c. Warm the solution in a water bath. Record observations.

EXPERIMENT NO. 9
ANALYSIS OF MILK

Group No. Name Course, Year & Section Date Score

TEST FOR FAT

OBSERVATION/RESULT (miscibility)
Precipitate and ethanol

TEST FOR CASEIN

OBSERVATION/RESULT
Precipitate solution

TEST LACTALBUMIN AND LACTOGLOBULIN

OBSERVATION/RESULT PRECIPITATION TEST
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(BIURET TEST) WITH HEAT AND ACETIC

ACID
MILK

TEST FOR LACTOSE

OBSERVATION/RESULT OBSERVATION/RESULT
(BENEDICT TEST) (BARFOED’S TEST
MILK

TEST FOR CALCIUM

OBSERVATION/RESULT
Milk

TEST FOR INORGANIC PHOSPHATE

OBSERVATION/RESULT
Milk