ExperimentT Proteins and Amino Acids
T-A. Protein Denaturation
T-C Electrophoresis of Amino Acids (Dry Lab)
Abstract:
@When we make a boiled egg, we find that an egg white in a row egg is clear and a cooked egg white is gwhiteh literally. This is the very example showing the denaturation of proteins. In this experiment, I watched the denaturation, which the structure of proteins is altered by chemical or physical reasons. The reagent chemically affecting the sample protein, Bovine Serum Albumin, was Nitric acid, Ethanol, Silver nitric acid, Trichloroacetic acid and the physical factor was heat. After adding each factor in order to denaturate the protein, each of them showed the alternation because of the reaction with the particular bonds and groups in the sample protein.

Introduction
@The structure of proteins can be classified into four levels: primary, secondary, tertiary, quaternary. The basic structure, primary structure, is made up with covalent bond which is the strongest binding in the chemical bonds, so the primary structure is hardly affected even if the protein changes its form. When the conformation of proteins is altered, this process is called denaturation, whereas the intact protein is referred to the native protein. Secondary, tertiary, quaternary structures were made up of weaker bindings than covalent bonds. Therefore, the high plane structures above primary structure are disrupted easily. This alternation results in denaturation and loses of the biological activity. The factors of denaturation are heat, strong acid, organic compounds, and heavy metal ions. Each sources of denaturation reacts on different bonds in the protein and makes the protein visible as a precipitate or coagulation. Through this experiment, we observed the alternation by means of applying heat and several reagents to the protein solution.

Materials and Methods
Materials:
@2% BSA (Bovine serum albumin), Nitric acid, 95% Alcohol,
@5% Silver nitric acid, 5% Trichloroacetic acid.
Methods:
A. Denaturation of proteins by heat, strong acids, or alcohol
@@1. Boil 3ml of a 2% BSA solution for a few minutes
@@2. Add 3ml of water to 1ml of 2% BSA solution. Pour this solution into a tube@containing nitric acid so as to form two layers.
@@3. Add 5ml of 95% ethanol to 3ml of 2% BSA solution, shake well.
B. Denaturation of proteins by heavy metal ions
@@1. Set up a test tube containing 3ml of 2% BSA. Add 5 drops of 5% silver nitrate. Shake the tube well.
C. Denaturation of proteins by organic acids
@@1. To a test tube containing 3ml of 2% BSA, add 1-2 ml 5% Trichloroacetic acid. Shake the tube well.

Results
T-A. Protein Denaturation
@A-1 As soon as the test tube was put into boiling water, the color of 2% BSA solution was changed into white. Few minutes later, several big white coagulations were formed like cottage cheese.
@A-2 Pouring nitrate quietly, two layers came in the test tube. The upper layer looked as white curd; the lower ones changed its color into pale yellow. After shaking, the color of solution changed into deeper yellow and small precipitation was floated in the solution.
@A-3 The solution was milky, but after several minutes, some big coagulation could be seen.
@B-1 As soon as I dropped silver nitrate in the test tube, the solution turned into white. In this test tube, the precipitation was really small so that I could not see that figure.
@C-1 The solution changed into white liquid.

T-C Electrophoresis of Amino Acids (Dry Lab)
Electrophoresis at pH 6
Amino acid -COO- -NH3+ -side chain pI direction
Alanine 2.3 9.9 --- 6.1 Stay original
Aspartic
Acid 2.0 10.0 3.9 2.95 Anode
Glutamic
Acid 2.2 9.7 4.3 3.25 Anode
Histidine 1.8 9.2 6.0 7.6 Cathode
Lysine 2.2 9.2 10.8 10 Cathode
<Predominant ionic form & charge>

Discussion
@In the experimentT-A, I watched the denaturation of protein with heat, strong acid and ethanol. Heat disrupts the bond in BSA, such as hydrogen bond and dispersion forces. Therefore, the hydrophobic side chains were no longer folded inside the protein and BSA was separated from the solution. Strong acid, like nitric acid, and ethanol break hydrogen bonds as well. Not like in the solution with ethanol, the precipitation in the nitrate solution was very fine and pale yellow. On the contrary, the precipitation of the ethanol solution was big and white. All three of the denaturation written above is responsible for the alternation of the secondary, tertiary, quaternary structure in BSA.

@The experimentT-B showed the denaturation by heavy metal ions. As heavy metal ions bond to carboxyl groups (-COO-) in BSA, which is stronger binds than that binding with positively charged side chain (NH3+), the tertiary structure was altered. This alternation might occur wherever carboxyl groups exist and resulted in the precipitate from the solution. Trichloroacetic acid reacts with negatively charged groups contained in aspartic acid and glutamic acid. This type of denaturation could be observed in the experimentT-C. How the structure of BSA was changed was same as the effect of heavy metal ions, but as written above, the part of group participating to the denaturation in the experimentT-C differ from the experimentT-B.

@Through the all experiments, I could see how the denaturation occurred and think which part of proteins takes part in this reaction. Because the denaturation of proteins is taken advantaged of in the variety area of food manufacturing and even in our cooking scene, it is important for us to understand what the denaturation is.
Answers for questions

Experiment 1-A
For each reagent (EtOH HNO3, and heat)
a) Name the type of linkage which is disrupted.
EtOH: Hydrogen bond
HNO3: Hydrogen bond
Heat: Hydrogen bond and Dispersion forces
b) Write on equation.

Experiment 1-B
1. Explain the change in terms of bonds and levels of structure disrupted.
@The heavy metal ions form strong bonds with the carboxyl groups. It means that the heavy metal ions disrupt the ionic bonds which connect with carboxyl group and another negatively charged group in the high place structure. As the linkages between the two -helix chains are broken, the tertiary structure may be altered.

2. Name 2 amino acids whose side chains may contribute ?COO- group.
Aspartic acid, Glutamic acid
3. Write a representative reaction showing the disruption of an ionic linkage using specific amino acid gRh groups.

Experiment 1-C
1. Explain the change in terms of bonds and levels of structure disrupted.
@Trichloroacetic acid reacts with positively charged side chains provided from histidine, arginine, and lysine. Usually, charged side chains are outside of proteins and take part in making the tertiary structure. If this bond is broken, the tertiary structure will be altered.
3. Name 3 amino acids whose side chains may contribute positively-charged groups.
Lysine, Arginine, Histidine.
2. Write an equation showing this disruption.
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