EXPERIMENT 2 Amino Acids BACKGROUND The purpose of this experiment is to become familiar with the dual nature of amino acids. Amino acids are unique in that they can exist in a zwitterion form. This separation of charge allows them to function both as a proton acceptor and as a proton donor at a specific ph known as the isoelectric ph or the pi. The ability of amino acids to exist in this form allows them to perform essential tasks. One such task is their ability to aid in buffering the blood in organisms. Since the blood stream is a short-term reservoir for amino acids (also known as the amino acid pool), their presence allows them to act as either a proton acceptor or as a proton donor. An amino acid is said to be in zwitterion form when the overall charge of the amino acid is zero. When the overall charge is zero, the amino acid is said to be electrically neutral. Since amino acids are the monomeric units of proteins, the side chain residues can collectively contribute to the overall pi of the protein. When amino acids are linked in a protein the α-amino group and the carboxyl group of every amino acid do not contribute to the overall charge of the protein because they are tied up in peptide bonds. In intact proteins only the side chain residues and the two peptide ends contribute to the overall charge of the protein. When a protein has an overall charge of zero (i.e. the negative charges balance the positive charges) the ph at which this occurs is referred to as the isoelectric ph (pi) of the protein. O + H 3 N-CH-C-O - R Zwitterion Form (Net charge is zero) The pk a values of side chain residues are important in the functions of proteins. The catalytic portions of proteins, also referred to as active sites, tend to contain many charged amino acid residues that participate in the reactions being catalyzed. There are two types of amino acids that under certain ph ranges are charged. Acidic amino acid residues are negatively charged at physiological ph, while basic amino acid residues are positively charged at physiological ph. In this experiment, the amino acid glycine will be titrated and the pk a values of its acidic protons will be determined. In addition, an unknown amino acid will also be analyzed by titration and, using the observed pk a values, the unknown will be identified. Finally, the ph dependence of the highly abundant milk protein, casein will be studied. Milk will be titrated until the pi of casein is reached. Afterward the the milk will be lowered to a ~1.0 to simulate the effect gastric juice has on protein during digestion.
PROCEDURE Part A: Titration of glycine with a base A solution of 1.0 M glycine will be titrated with 1.0 M NaOH. The solution of glycine has been pre-acidified, therefore the amino acid glycine should be in its fully protonated form. Using a graduated cylinder add 30 ml of 1.0 M glycine to a 100 ml beaker. Add a magnetic stir bar and place the beaker on a stir plate. Start the stirring at a medium speed. Wash the ph meter electrode with distilled water several times and then gently wipe the electrode dry with a Kimwipe. Calibrate the ph meter with the three standard calibration solutions the ph 4.0 standard, the ph 10.0 standard, and finally the ph7.0 standard. After calibration thoroughly wash the electrode with distilled water and dry the excess water with a Kimwipe. Then immerse the electrode into the glycine solution making sure that the tip of the electrode is covered by solution, but not so deep that the stir bar will hit the electrode. Record the initial the glycine solution. Slowly add 0.5 ml increments of 1.0 M NaOH and record the the solution after each addition in Table I. Remember to allow the ph to stabilize before adding the next aliquot of base. After the titration is complete wash the ph meter electrode with distilled water and dry the excess water with a Kimwipe. Plot the data obtained to determine the pk a of the two acidic protons on glycine and determine the isoelectric point (pi). The ph values should be plotted on the y-axis, while the volume of base added should be plotted on the x-axis. TABLE I: TITRATION OF GLYCINE 0.0 ml 22.0 ml 44.0 ml 0.5 ml 22.5 ml 44.5 ml 1.0 ml 23.0 ml 45.0 ml 1.5 ml 23.5 ml 45.5 ml 2.0 ml 24.0 ml 46.0 ml 2.5 ml 24.5 ml 46.5 ml 3.0 ml 25.0 ml 47.0 ml 3.5 ml 25.5 ml 47.5 ml 4.0 ml 26.0 ml 48.0 ml 4.5 ml 26.5 ml 48.5 ml 5.0 ml 27.0 ml 49.0 ml 5.5 ml 27.5 ml 49.5 ml 6.0 ml 28.0 ml 50.0 ml 6.5 ml 28.5 ml 50.5 ml 7.0 ml 29.0 ml 51.0 ml 7.5 ml 29.5 ml 51.5 ml 8.0 ml 30.0 ml 52.0 ml 8.5 ml 30.5 ml 52.5 ml 9.0 ml 31.0 ml 53.0 ml 9.5 ml 31.5 ml 53.5 ml
10.0 ml 32.0 ml 54.0 ml 10.5 ml 32.5 ml 54.5 ml 11.0 ml 33.0 ml 55.0 ml 11.5 ml 33.5 ml 55.5 ml 12.0 ml 34.0 ml 56.0 ml 12.5 ml 34.5 ml 56.5 ml 13.0 ml 35.0 ml 57.0 ml 13.5 ml 35.5 ml 57.5 ml 14.0 ml 36.0 ml 58.0 ml 14.5 ml 36.5 ml 58.5 ml 15.0 ml 37.0 ml 59.0 ml 15.5 ml 37.5 ml 59.5 ml 16.0 ml 38.0 ml 60.0 ml 16.5 ml 38.5 ml 60.5 ml 17.0 ml 39.0 ml 61.0 ml 17.5 ml 39.5 ml 61.5 ml 18.0 ml 40.0 ml 62.0 ml 18.5 ml 40.5 ml 62.5 ml 19.0 ml 41.0 ml 63.0 ml 19.5 ml 41.5 ml 63.5 ml 20.0 ml 42.0 ml 64.0 ml 20.5 ml 42.5 ml 64.5 ml 21.0 ml 43.0 ml 65.0 ml 21.5 ml 43.5 ml Part B: Identification of an unknown amino acid based on observed pk a values In this part of the experiment you will titrate an unknown amino acid. The amino acid has been pre-acidified so that it is in its fully protonated form. The amino acid solution is a 1.0 M solution and 1.0 M NaOH will again be used to titrate the amino acid. Use a graduated cylinder to add 30 ml of the unknown 1.0 M solution to a 150 ml beaker. Add a magnetic stir bar to the beaker and place the beaker of solution on a stir plate. Stir the solution at a medium rate. Wash the ph meter electrode several times with distilled water and dry the excess water with a Kimwipe. Record the initial the unknown solution. Then add 0.5 ml increments of 1.0 M NaOH, until 40 ml of base has been added. The final 40 ml of base required to complete the titration will be added in 1.0 ml increments. After each increment record the ph measurement in Table II. In your analysis of the data determine the pk a values of the acidic protons in the unknown amino acid and the pi. The ph values should be plotted on the y-axis, while the volume of base added should be plotted on the x-axis.
TABLE II: TITRATION OF UNKNOWN AMINO ACID 0.0 ml 20.5 ml 42.0 ml 0.5 ml 21.0 ml 43.0 ml 1.0 ml 21.5 ml 44.0 ml 1.5 ml 22.0 ml 45.0 ml 2.0 ml 22.5 ml 46.0 ml 2.5 ml 23.0 ml 47.0 ml 3.0 ml 23.5 ml 48.0 ml 3.5 ml 24.0 ml 49.0 ml 4.0 ml 24.5 ml 50.0 ml 4.5 ml 25.0 ml 51.0 ml 5.0 ml 25.5 ml 52.0 ml 5.5 ml 26.0 ml 53.0 ml 6.0 ml 26.5 ml 54.0 ml 6.5 ml 27.0 ml 55.0 ml 7.0 ml 27.5 ml 56.0 ml 7.5 ml 28.0 ml 57.0 ml 8.0 ml 28.5 ml 58.0 ml 8.5 ml 29.0 ml 59.0 ml 9.0 ml 29.5 ml 60.0 ml 9.5 ml 30.0 ml 61.0 ml 10.0 ml 30.5 ml 62.0 ml 10.5 ml 31.0 ml 63.0 ml 11.0 ml 31.5 ml 64.0 ml 11.5 ml 32.0 ml 65.0 ml 12.0 ml 32.5 ml 66.0 ml 12.5 ml 33.0 ml 67.0 ml 13.0 ml 33.5 ml 68.0 ml 13.5 ml 34.0 ml 69.0 ml 14.0 ml 34.5 ml 70.0 ml 14.5 ml 35.0 ml 71.0 ml 15.0 ml 35.5 ml 72.0 ml 15.5 ml 36.0 ml 73.0 ml 16.0 ml 36.5 ml 74.0 ml 16.5 ml 37.0 ml 75.0 ml 17.0 ml 37.5 ml 76.0 ml 17.5 ml 38.0 ml 77.0 ml 18.0 ml 38.5 ml 78.0 ml 18.5 ml 39.0 ml 79.0 ml
19.0 ml 39.5 ml 80.0 ml 19.5 ml 40.0 ml 20.0 ml 41.0 ml Part C: Observing the effects of ph changes on the milk protein casein In this part of the experiment you will be determining the effect of ph changes on an entire protein, specifically casein. Casein is an abundant protein in bovine milk, constituting approximately 80% by weight of the total protein. There are actually three types of casein proteins in bovine milk named α, β, and κ. They are called phosphoproteins because they have phosphoryl groups covalently bound to serine and threonine side chains. Addition of excess acid causes casein to denature and form a precipitate. Measure 25 ml of low fat milk and transfer it to a 25 ml plastic test tube. Measure the initial the milk using the ph meter. Then slowly add 1.0 ml increments of 0.5 M HCl to the milk until protein begins to aggregate. Invert the falcon tube several of times after each 1.0 ml increment is added to help mix the solution. Measure the the milk solution after aggregates are observed. Indicate the ph value in Table II along with your observations and the total amount of acid added. Then continue adding 1.0 ml increments of 0.5 M HCl to the milk until a 1.0 is reached. Write down your observations in Table III. TABLE III Acid Added (ml) 0.0 ml the Milk Observations Additional Questions 1. Determine the pi of glycine using the titration curve constructed with your data. Determine the percent error in your experimental pi value. The established pi value for glycine can be found in any biochemistry book.
2. Identify the unknown amino acid based on your experimental pk a values and information obtained in the literature. Using your titration curve determine the isoelectric ph for the unknown amino acid. Calculate the percent error in your experimental value using the accepted isoelectric ph value for this amino acid. Comment on why this amino acid would be useful to have in the active site of an enzyme. 3. Comment on what you think is occurring with the protein in milk at the two different stages of the experiment. Why is the ph dependence of protein in milk important for people in the food industry to understand?