Protein Analysis
Proteins are complex, specialized molecules of sequences of amino acids. There are twenty different amino acids commonly found
in proteins. All of these amino acids have a similar structure. At the center of the molecule is the alpha carbon. Except for the
simpliest amino acid, glycine, all amino acids have chiral centers and optical isomers. The alpha carbon is bonded to four different
groups: (1) an amino group (NH2), (2) a carboxyl group (COOH), (3) a hydrogen atom and (4) the R group or side chain. . The
different amino acids have different R groups consisting of a carbon chain and in many cases other carboxyl groups, amino groups
or other functional groups
Amino acids are linked together by condensing the amino group of one amino acid with the carboxyl group of another. The amino
acids are linked together by peptide bonds formed when the carboxyl group of one amino acid reacts with the amino group of the
next amino acid in a dehydration synthesis reaction. These long unbranched chains of amino acids, typically 10 to 60 amino acids in
length, are known as a polypeptides. . As each amino acid is added to the growing polypeptide chain, a molecule of water is formed.
Polypeptides have an unreacted carboxyl group at one end of the molecule (known as the C-terminus).and an unreacted amino
group at the other end of the molecule (known as the N-terminus) Chemists describe the structure of a specific polypeptide by
writing down the sequence of amino acids starting at the N-terminus and proceeding along the chain to C terminus. The difference
between a polypeptide and a protein is partly a matter of chain length. Chains of amino acids more than 60 amino acids in length
are regarded as proteins. Proteins may also have more than one polypeptide chain bonded together.
Proteins play important roles in living organisms. Structural proteins such as collagen, provide support. Regulatory proteins control
cell processes. Storage proteins produced in reproductive structures are a source of amino acids for developing organisms, e.g.,
casein in milk or albumin. Contractile proteins are responsible for movement of cells and organisms. Transport proteins carry
substances from one place to another, e.g., hemoglobin carries oxygen throughout the human body. Proteins also serve as
antibodies, hormones, receptors, and enzymes.
For this experiment we will conduct various tests on proteins extracted from egs and milk. The egg white is rich in albumin although
it contains water, lipids, and other carbohydrates as well. However they generally do not interfer with the protein tests that you will
be doing. Caesin from milk must be extracted. A procedure for doing so is discussed below
Isolation of Casein from Milk
Gently warm 20 ml of skim milk in a 100-ml beaker on a hot plate. Once the milk has reached a temperature of about 40-50
degrees, remove the beaker from the heat and, while stirring with a glass rod, add 2 ml 10% aqueous acetic acid solution along with
2 ml 1.0 M sodium acetate. Stir the suspension to break up the solid or curd (casein and any residual fat) into small pieces the size
of rice grains. Split the suspension into two centrifuge tubes and centrifuge for about 45 seconds. The solids at the bottom of the
tube are casein protein
Protein and amino acid tests
1. Biruet Test: The biuret test will indicate the presence of amino acid residues of peptides containing two or more amino acid
residues and therefore is used to determine whether or not a protein is present. This test relies on the fact that amino acid residues
form a colored complex with Cu+2 ion in basic medium:
All proteins should give a positive test whereas simple amino acids should give a negative test. Biuret reagent is a light blue solution
which turns purple when mixed with a solution containing protein. The purple color is formed when copper ions in the biuret reagent
react with the peptide bonds of the polypeptide chains to form a complex.
1. Add 1 mL (20 drops) of sample to each tube.
2. Add 1 mL of biuret reagent to each tube.
3. Mix the contents of each tube using a stirring rod
4. Wait 2 minutes.
5. Examine each tube carefully. Note the color.
6. Record your observations in the Table. Indicate presence (+) or absence (-) of peptide bonds in each sample.
Ninhydrin Test for Amino Acids: Because amino acids contain a free amino group, they are readily detected with ninhydrin
reagent which reacts with free amino groups to form a purple or violet colored substance. Ninhydrin reagent can also be used to
detect proteins, but the proteins must be heated or digested to hydrolyze the protein into free amino acids. When heated with
ninhydrin, amino acids give characteristic deep blue colours (or occasionally pale yellow). The reactions involved in this test are
shown below.
1. Put a drop of each sample on a piece of filter paper. Draw a circle around the spot with a soft pencil and write the
name of the sample next to the spot.
2. Allow all spots to dry thoroughly.
3. Put a drop of ninhydrin reagent on each spot. Wait for at least 20 minutes. Observe each spot carefully. Record your
observations. In paticular make note of the color and the intensity of color A color change indicates the. Indicate
presence of free amino groups, either in the form of amino acids or as side chains in a polypeptide
Xanthoproteic Acid Test: This test will determine if residues of tyrosine or tryptophan are present. The solution to be tested is
treated with concentrated nitric acid, which will nitrate the benzene rings of those residues. The nitrated aromatic rings are yellow in
color and are called Xanthoproteic acids (Xantho, = yellow, Greek). If base is added, the color becomes more intense and the color
will shift more to orange. The reaction is illustrated for a tyrosine residue.
Add 1 mL of concentrated nitric acid to 2 mL of the protein solution to be tested in a test tube. Note the appearance of any heavy
white precipitate. Warm the solution carefully in a hot water bath, noting any change to a yellow-colored solution. Cool the test tube
in a stream of cold water and carefully add 10% NaOH with gentle agitation. Note if the color deepens to orange, which would be a
positive test.
Sulfur Test The presence of sulfur-containing amino acids such as cysteine can be determined by converting the sulfur to an
inorganic sulfide through cleavage by base. When the resulting solution is combined with lead acetate, a black precipitate of
lead sulfide results.
Sulfur-containing protein ----NaOH----> S2- ----Pb2+----> PbS
Place 1 mL of the protein solution in a 16 x 150 mm test tubes. Add 2 mL of 10% aqueous sodium hydroxide. Add 5 drops of
10% lead acetate solution. Stopper the tubes and shake them, then remove the stoppers and heat in a boiling water bath for 5
minutes. Cool and record the results.