Download Pre-lab 8: Peptides and Proteins

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Pre-lab 8: Peptides and Proteins
Read the background information and answer the following question before lab.
1. Draw the zwitterion of the amino acid, Valine.
2. Draw the zwitterions of the amino acid, phenylalanine
3. Draw one of the dipeptides that can form from Valine and Phenylalanine.
4. Which types of structures are disrupted by the denaturing of a protein?
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Lab 8: Peptides and Proteins
Objective: The objectives of this experiment are to observe the denaturing of proteins, use the
isoelectric point of casein in milk to isolate the protein, and use a chemical test to identify
proteins and peptides (tripeptides and higher).
Background Information:
Peptide bonds:
A dipeptide forms when a two amino acids bond together. A peptide (or amide) bond forms
between the carboxylic acid of one amino acid and the amino group of the next amino acid with
the loss of H2O. A representation of this is shown in Figure 1.
Figure 1.
Structure of Proteins:
When many amino acids are joined by peptide bonds they make a polypeptide. If more than
50 amino acids are in the peptide chain, it is usually considered to be a protein. Proteins make
up many important features in the body including skin, muscle, cartilage, hair, fingernails,
enzymes, and hormones. Proteins have specific structures, which are determined by the
sequence of the amino acids. The peptide bonds that join one amino acid to the next are the
primary level of protein structure. Secondary structures include the alpha helix formed by the
coiling of the peptide chain, or a β–pleated sheet structure formed between protein strands. The
structures are shown in Figure 2.
The secondary structures are held in place by many hydrogen bonds between the oxygen
atoms of the carbonyl groups and the hydrogen atoms of the amide group. At the tertiary level,
interactions between the side groups such as ionic bonds or salt bridges, disulfide bonds, and
hydrophilic bonds give the protein a compact shape. Similar interactions between two or more
tertiary units produce the quaternary structure of many active proteins.
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Figure 2.
Denaturing of Proteins:
Denaturing of a protein occurs when certain conditions or agents disrupt the bonds that hold
together the secondary or tertiary structures of a protein. Proteins are denatured with heat, acid,
base, ethanol, tannic acid, and heavy metal ions (silver, lead, and mercury). In most cases, the
protein coagulates. Heat increases the motion of the atoms and disrupts the ionic bonds between
amino acids with the acidic and basic side groups. Alcohol, an organic solvent, destroys
hydrogen bonds. The heavy metal ions, Ag+, Pb2+, and Hg2+, reacts with the sulfur groups and
carboxylic acid groups, which prevents the formation of the cross-links for tertiary structures and
quaternary structures.
Isolation of Casein (Milk Protein):
A typical source of protein is milk, which contains the protein casein, When the pH of a
sample of nonfat milk is acidified, it reaches its isoelectric point, and the protein separates out of
the solution. The change in pH disrupts the bonds that hold the tertiary structure together.
Adjusting the pH of the mixture causes the casein to solidify so it can be removed. A similar
process is used in the making of yogurt, cheeses, and cottage cheese. An enzyme provides the
acid for the denaturing of the protein for those products. In this experiment, the mass of a
quantity of milk and the mass of isolated casein will be determined. From the data, the percent
casein in milk will be calculated.
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Color Tests for Proteins:
Certain tests give color products with amino acids, peptides, and/or proteins. The results of
the test can be used to detect certain groups or type of bonds within proteins or amino acids.
Biuret test The biuret test is positive for a peptide or protein with two or more peptide bonds. In
the biuret test, the blue color of a basic solution of Cu2+ turns to a violet color when a tripeptide
or larger peptide is present. Individual amino acids and dipeptides do not react with the reagent,
and the solution will remain blue (negative).
Procedure/data:
Part I. Denaturing of Proteins.
Equipment list:
 5 Test Tubes
 Test Tube Holders
 10 mL Graduated Cylinder
Place 2-3 mL of egg albumin solution in each of five test tubes. Use one sample for each of the
following tests. Record your results on the chart.
1. Heat. Using a test tube holder, heat the egg albumin solution over a low flame. Describe any
changes in the solution.
2. Acid. Add 2 mL of 10% HNO3.
3. Base. Add 2 mL of 10% NaOH.
4. Alcohol. Add 4 mL of 95% ethanol. Mix.
5. Heavy metal ions. Add 10 drops of 1% AgNO3.
Number: Change in the Solution
1
2
3
4
5
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Part II. Isolation of Casein (Milk Protein).
Equipment list:
 150 mL Beaker
 Hot Plate
 Thermometer
 Funnel
 Filter Paper
 Watch Glass
 pH Paper
 Stirring Rod
1. Weigh a 150 mL beaker. Add about 20 mL of nonfat milk to the beaker and weigh.
2. Using pH paper, determine the pH of the milk sample.
3. Warm the sample on a hot plate until the temperature reaches about 50oC. Remove the
beaker and milk from the heat and add 10% acetic acid, drop by drop. You may need 2-3
mL. Stir continuously. At the isoelectric point, the casein (milk protein) becomes insoluble.
When no further precipitation occurs, stop adding acid. If the liquid layer is not clear, heat
the mixture gently for a few more minutes. Determine the pH at which the casein becomes
insoluble in solution. This is the pH of the isoelectric point of casein.
4. Collect the solid protein using a funnel and filter paper. Wash the protein with two 10 mL
portions of water. Weigh a watch glass. Transfer the protein to the watch glass and let the
protein dry. Weigh. Calculate the mass of the milk protein. Save for part E.
5. Calculate the percentage of casein in the nonfat milk.
% casein = (mass in grams of casein/ mass in grams of milk) X 100%
Weight of Beaker and Milk
Weight of Beaker
Weight of Milk
pH of Milk
Isoelectric Point of Casein
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Weight of Watch Glass and Casein
Weight of the Watch Glass
Weight of Casein
% Casein
Part III. Color Test for Protein.
Equipment list:
 Tests Tubes
 Test Tube Rack
 Spatula
Biuret Test.
1. In four separate test tubes, place 2 mL of solutions of glycine, tyrosine, gelatin, and agg
albumin. To the fifth tube, add a small amount of the solid casein (from part D), the amount
held on the tip of a spatula.
2. To each sample, add 2 mL of 10% NaOH and stir. Then add 5 drops of biruet reagent (5%
CuSO4), and stir. Record the color of each sample. The formation of a pink-violet color
indicates the presence of a protein with two or more peptide bonds. If such a protein is not
present, the blue color of the cupric sulfate will remain (negative result). Record the results
on the chart.
Compound:
color
Gylcine
Tyrosine
Gelatin
Egg Albumin
Casein
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Name:_______________________
Partner(s):____________________
Lab 8: Peptides and Proteins
_____________________________
Section:______________________
Part I. Denaturing of Proteins.
Number: Change in the Solution
1
2
3
4
5
Part II. Isolation of Casein (Milk Protein).
Weight of Milk
pH of Milk
Isoelectric Point of Casein
Weight of Casein
% Casein
Part III. Color Test for Protein.
Biuret Test.
1. Which Compounds where positive for the biuret test?
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