Download Lab Protein and Amino Acids

Lab Protein and Amino Acids
Objectives:
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

Observe chemical behavior of amino acids and proteins
Qualitatively analyze amino acids and proteins
Isolate protein from natural source
Background:
The term protein was first used by Gerardus Mulder in 1838 to describe the complex nitrogencontaining organic compounds that are found in all living cells. It is derived from the Greek word
proteios, which means “of first importance.” This is an appropriate description of these important
compounds since proteins are involved in essentially all biochemical processes in the body.
Living cells in both plants and animals are approximately 60 % water and 40% solid material,
roughly half of which is protein. In plants, proteins are synthesized from carbon dioxide, water,
nitrates, sulfates, and smaller amounts of several other compounds. Animals, on the other hand,
are not able to synthesize proteins in this way, so proteinaceous material must be obtained in
the diet.
Most protein in the body is used in body building and repair. The principal component of all
enzymes is protein. While lipids and, to a lesser extent, carbohydrates are stored in the body
ads an energy reserve, the storage of protein is almost nonexistent. Protein is little used as a
source of energy. Consequently, for good health, it is necessary to have a regular intake of
protein through the diet. An animal can survive for a limited time on a diet that contains only
vitamins, minerals, and proteins (no carbohydrates or lipids). But if the animal is fed a diet
containing everything but protein, premature death will follow.
Amino Acids
Proteins are polymers of amino acids. For the most part, proteins are very large molecules
composed of hundreds of amino acid units. Proteins can be broken known into the amino acids
that compose them by hydrolysis (reaction with water). In the laboratory, hydrolysis reactions
are usually carried out in the presence of strong acids (HCl or H2SO4), strong bases (NaOH or
KOH) or proteolytic enzymes called proteases. Amino acids are carboxylic acids that contain an
amino acid group, -NH2, joined to the alpha-carbon of the acid. There are twenty amino acids
normally found in nature. The alpha position in a carboxylic acid is the carbon atom adjacent to
the carboxyl group. An alpha-amino acid has an amino acid group attached to the alpha-carbon
atom.
O
H O
C C OH
the alpha carbon
H2N C C OH
R
an alpha-amino acid
All alpha amino acids can be represented by the same general formula. The only difference
between one amino acid and another is the nature of the R group bonded to the alpha-carbon
atom. The R group of an amino acid can be hydrogen, a simple alkyl group, a group containing
an aromatic ring, a heterocyclic group or other substituted group. Each different amino acid has
a unique set of properties due to the composition of its R group.
H H
the alpha carbon
H N C COOH
R The R substituent is different
for each amino acid.
Amino acids are commonly classified in terms of the number of carboxyl, -COOH, and amino, NH2, groups in the molecule. Neutral amino acids contain one carboxyl group and one amino
group. Aqueous solutions of neutral amino acids have a neutral or near neutral pH (~7). Basic
amino acids contain one carboxyl group but more than one amino or amino-like group. Aqueous
solutions of basic amino acids have a basic pH (>7). Acidic amino acids contain one amino
group but more than one carboxylic acid group. Aqueous solutions of acidic amino acids have
an acidic pH (<7).
Since -amino acids contain both acidic -COOH and the basic –NH2 groups. Unfortunately,
though, the picture is not as simple as this. In the solid crystalline state the a-amino acids
exist as zwitterions, as discussed before they are formed by the transfer of protons, H+ from
the -COOH to the –NH2 groups. For -amino acids without acidic or basic side chains these
zwitter ions have charged groups but are neutral overall. This is shown below.
H O
+
C C O
NH3
R
Zwitter ions remain when the -amino acid is dissolved in water at pH 7. Addition of an acid,
supplying more protons, produces ions with an overall positive charge. The amino acid
forms the below structure in an acid environment.
H O
H O
+
NH3
C C O
+
H
+
NH3
C C OH
R
R
Addition of a base, removes the acidic hydrogens, producing ions with an overall negative
charge. The amino acid forms the below structure in a basic environment.
H O
H O
+
NH3
C C O
R
-
OH
NH2
C C O
R
Peptides
As was stated earlier, the complete hydrolysis of a protein will yield a mixture of amino acids.
But if the hydrolysis is stopped before the protein is completely broken down, small fragments
composed of only a few amino acid units can be isolated. These small fragments are called
peptides. Each peptide will contain one or more amide unit commonly called a peptide link. A
peptide link forms when amino acids join to form the protein.
When two amino acids combine, the amino group of one amino acid reacts with the carboxyl
group of the second to form water and a dipeptide (a peptide made up of two amino acid units).
By convention, amino acids are written in a general equation with the amino groups to the left
and the carboxyl groups to the right.
NH3
H O
H O
C C NH
C C O
CH3
H
If amino acid 1 and amino acid 2 react in the reverse order, a different dipeptide, an isomer of
dipeptide A, results. The isomer, which we will call dipeptide B, is shown below. A third amino
acid could join with either dipeptide to form a tripeptide, and the peptide could continue to grow
in this way, also shown below.
H O
H O
NH3
NH3
C C NH
C C O
CH3
H
H O
H O
H O
C C NH
C C NH
C C O
CH3
H
CH2
OH
Biochemists have developed a shorthand method for describing the composition of peptides
using the abbreviation of the amino acids. The amino groups are understood to be on the left
and the carboxyl groups on the right. The formation of a tripeptide from glycine (gly or G),
alanine (ala or A), and serine (ser or S), in that order, would be written this way:
gly-ala-ser
or
GAS
Two test reagents will be used in this lab, Ninhydrin and Biuret. For the Biuret test, a positive
test for peptide bonds is the blue reagent turns violet in the presence of proteins and pink color
persists with short-chain polypeptides. The color intensity varies with concentration of peptides.
The Biuret Reagent is made of sodium hydroxide and copper sulfate.
For the Ninhydrin Test a positive test for amino acids, not peptides, gives a give blue color,
except proline which gives a yellow color.
Procedure:
Part 1 pH:
1. Label four clean test tubes 1-4
2. Add the following:
a. Tube 1: 5 drops 1% asparagine
b. Tube 2: 5 drops 1% alanine
c. Tube 3: 5 drops 1% arginine
d. Tube 4: 5 drops 1% aspartic acid
3. Determine the pH of each solution
Part 2 Amino Acid Buffers:
1. Label four clean test tubes 1-4
2. Add the following:
a. Tube 1: 2 ml DI water
b. Tube 2: 2 ml DI water
c. Tube 3: 2 ml 4% glycine
d. Tube 4: 2 ml 4% glycine
3. Determine pH of each tube
4. Add the following:
a. Tube 1: 5 drops .1 M HCl
b. Tube 2: 5 drops .1 M NaOH
c. Tube 3: 5 drops .1 M HCl
d. Tube 4: 5 drops .1 M NaOH
5. Determine pH of each tube
Part 3 Isolation of Casein:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Add 20 ml of milk to an Erlenmeyer flask
Heat to 40C and add 5 drops of glacial acetic acid, stir for 1 minute.
Filter the solution thru a cheesecloth filled funnel, record observations.
Ring out the cloth and scrape the solid into a beaker.
Add 10 mL of 95% ethanol.
Break up the solid in the solution, decant the liquid.
Add 10 mL of 1:1 ether-ethanol to the beaker.
Break up the solid in the solution.
Filter the solution thru a cheesecloth filled funnel, record observations.
Smear small amount of solid thinly on a paper towel.
On a different section of the same paper towel, drip several drops of 4% glycine.
Lightly spray both areas of the paper towel with Ninhydrin and record results.
Smear small amount of solid thinly on another paper towel.
On a different section of the same paper towel, drip several drops of 4% glycine.
Lightly spray both areas of the paper towel with Biuret reagent and record results.
Part 4 Denaturation of a Protein:
1.
2.
3.
4.
Label four clean test tubes 1-4
To each tube add a small amount of egg white.
Add the following:
a. Tube 1: 20 drops 95% ethanol
b. Tube 2: 3 drops 0.1 M AgNO3
c. Tube 3: 3 drops 0.1 M HgCl2
d. Tube 4: 3 drops 0.1 M Pb(CH3COO)2
Record all observations
Concluding Questions:
Describe the 4 amino acids in the pH test part as acidic, basic or neutral.
Did the glycine behave as a buffer, explain.
Did your analysis of the casein determine it to be an amino acid chain or free amino acids,
explain.
When a protein is denatured, what is occurring?
Rank the reagents in the denature portion of the lab, most destructive to least destructive.
Define the following terms that are associated with proteins and amino acids.
peptide link
amino acid
dipeptide