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Protein Biochemistry:
Evidence for Charged Amino Acids in Proteins

By David Masterman



Type of Activity:

  • Hands-on

Target Audience:

  • Advanced biology
  • AP biology
  • Biochemistry

Background Information

This lab/demo requires the use of computer interfacing equipment. The Vernier pH and light probes were used with Data Logger software in our classrooms.
  1. To prepare the casein solution dissolve 1.2 g casein into 250mL of 0.01M NaOH solution for each lab team. To do this, place a beaker with 0.01M NaOH and magnetic stir bar on a magnetic stirrer and stir at a moderate speed. Slowly sprinkle a small amount of purified casein powder onto the surface of the liquid. Repeat this until all of the casein is dissolved. If too much casein is added at one time, the protein may lump. If sufficient time is available, have each student team prepare their own solution.

  2. Calibrate the pH probes prior to the lab.

  3. Students must add the acid and base dropwise -- not in a stream -- as the protein precipitates. If either are added too rapidly, students may miss detail or the protein may not have sufficient time to renature. Casein does not buffer the solution well when it is insoluble.

  4. Instructors may consider doing this as a demonstration rather than as a laboratory exercise.

  5. Casein will normally only go through a maximum of 4 to 5 cycles. When the denatured protein renatures itself, molecules entangle. Eventually, the molecules will entangle so much that they will not be able to fully dissolve.

  6. Students should complete the analysis using Data Logger software.

Results

The following data will be different from student's results. The actual data depend upon the rate of adding acid and base, among other things. Common errors that occur are:
  • The vortex interferes with the light sensor's readings.
  • Students do not add the acid and base dropwise as the protein precipitates.

In this window, the first of six cycles is highlighted and analyzed. A precipitate, indicated by low light intensity, forms at approximately pH 5-4-3.5. The protein is least soluble at pH 4.7. The number of positive charges on casein appears to be equal to the number of negative charges when the solubility of casein is between 4.2 and 5-3. This corresponds nicely with the pH of 4.7 used to separate casein from milk. When only one type of charge exists on casein, it will be soluble. Similar charges tend to repel each other -- this repulsion prevents molecules from attracting each other and forming a precipitate. At acidic pH's, below 3.5, only positive charges are present. At basic pH's, above 5.4 in the first cycle, only negative charges are present.

This lab demonstrates not only the reversible denaturation of a protein mixture, but provides evidence for the existence of electrical charges on the protein. This lab is highly successful for the advanced student, as it visually allows one to explore this complex phenomenon. This lab was the result of a conversation with Dr. Jerry Bell at the Woodrow Wilson Chemistry Institute, 1983.


Abstract/Summary

A protein's shape and structure are in part determined by its balance of positive, negative, and neutral charges. Positively charged amino acids attract negatively charged ones. This might cause one region of a protein to "stick" to another region of the protein. If the pH varies substantially, the protein might reversibly denature, or unravel its structure. In this experiment, one will determine whether a purified solution of the protein casein will reversibly denature, and observe at which pH values the protein forms a precipitate. Since the precipitate is a white solid, it will cloud the solution and block light from entering a light sensor. A pH probe will be used to determine the pH of the mixture as acid is added or as base is added.

Procedure

  1. Obtain and calibrate a computer interfaced light sensor and a pH probe.
  2. Place casein solution (1.2g casein in 250 mL of 0.01M NaOH) into a 600-mL beaker.
  3. Position the pH probe, lamp, and the light meter as shown at the left and prepare a computer for data collection. Set the data collection program to measure pH and light intensity as a function of time. Three plots might be displayed:
    • pH vs. -time
    • light vs. time
    • light vs. pH.
    • Start measuring and record all observations.
  4. Add 1M HCI acid dropwise. A white precipitate will form and then dissolve as the solution becomes more acidic and the protein becomes positively charged. Make sure that the addition of acid is slowed when the solution starts to become cloudy. Continue adding acid until either the white precipitate dissolves or the pH is less than two units.
  5. Repeat this process in reverse by adding 1M NaOH dropwise until a white precipitate forms. Continue adding base until either the white precipitate dissolves or the pH exceeds 13.
  6. Repeat steps 5 and 6 several times, completing three or four cycles of adding HCI and NaOH. After a while, the protein will refuse to dissolve. At this point, do not add additional acid or base. Print the graph and save your data.

A wide variety of explorative questions may be asked about the behavior of the casein mixture in acidic and basic conditions. Included are the following:

  • What was the appearance of the mixture at different light intensity values? Different pH values?
  • Over what pH age is the protein least soluble? Most soluble?
  • What appears to be the relationship between the light intensity and the pH of the mixture?
  • Over what pH range is the protein insoluble? Soluble? How did you determine this?
  • Over what pH values does the number of positive charges on casein appear to be equal to the number of negative charges in the protein?
  • Over what pH range do only positive charges exist on casein? Negative charges?
  • What evidence do you have that the denaturation of casein is reversible?


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