Biology Lab Week 4

pH, Buffers, and Enzyme Activity

Announcement: Bring your biology lab notebooks to the afternoon session on Monday October 16.
Goals:

  • Continue characterization and examination of bacteria
  • Learn the correct use of electronic pH meters.
  • Review the chemistry of buffers.
  • Examine the effect of pH (and possibly other factors) on enzymatic activity.

Useful Reading:
Any general chemistry textbook on buffers and pH measurement. This is briefly discussed in Solomons pp. 104-06

Material on enzymes in MBOC pp. 162-163, 166-168
Preparation: Find the structures for Tris buffer, catechol, and benzoquinone. Translate the names of the phosphate salts used to the correct formulas.
Sigma buffer reference center :http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Buffer_Explorer/Key_Resources/Buffer_Reference_Center.html

Old Business:

  • Examine your Coliscan plates and interpret the results.
  • Another simple to use enzyme assay that can characterize bacteria is assays for catalase and peroxidase. The catalase test is simple-place a small amount of your test bacteria on a slide and add a drop of 3 % hydrogen peroxide to the edge. The degree of fizzing is associated with the amount of catalase present-some bacteria produce a great deal, some produce none. Try this on some known bacteria and some unknown colonies.


Introduction:
One of the most important solutions properties for any biological reaction is pH. Virtually all reactions in biochemistry and molecular biology are run in buffered solutions that set the initial pH of the solution and minimize changes in pH due to additions of acid or base. In this lab you will prepare some buffers commonly used in biochemistry and molecular biology. Then the activity of an enzyme will be examined at various values of pH. It is useful to think that there is a universe of several dimensions in which proteins can function in some range of each variable- pH, salt content, oxidation potential, temperature, for example. Here we will start characterizing the environmental space in which polyphenol oxidase (often called catechol oxidase) functions. A summary of the theory and use of pH meters is included as an appendix.
 
A pH buffer is a solution of a conjugate acid-base pair. A buffer works best when the concentrations of the acid and base components are nearly equal. This means that a buffer is most effective when the desired pH is near the pKa of the acid-base pair being used. Unfortunately, there are not a large number of common compound with pKa values near 7-8, the normal physiological range. A large number of synthetic buffers (Tris, HEPES, MOPS, and a range of other abbreviations; see the Sigma resource listed in the reading) have been developed to provide buffers with convenient pKa values and low reactivity with biological systems. (These are often referred to as “Good buffers” named for the scientist who discovered them. He was studying mitochondria and wanted to control pH without fixing the amount of phosphate present.) The effectiveness of a buffer also depends on its concentration; a more concentrated buffer is more resistant to acid-base change than a dilute buffer. In increasing a buffer’s strength, however, limitations due to having too high a salt content or solubility problems may be encountered.

For today's lab you will prepare a series of pH buffers using 2 acid-base pairs. After calibrating a pH meter, you will then measure the pH of the buffers prepared and use these values to estimate the pKa of the acid-base systems involved.  You will then use several of the prepared buffers and examine the effect on an enzymatic reaction rate of pH.

The enzyme used today is polyphenol oxidase isolated from potato or sweet potato. This enzyme oxidizes polyphenols to associated quinones using molecular oxygen as an oxidizing agent. Our test substrate is catechol, with the following balanced equation:


catechol + O2 → benzoquinone + water


This reaction is responsible for the commonly observed browning that occurs on many fruits, vegetables, and mushrooms upon being cut. A similar enzymatic activity produces the animal pigment melanin from tyrosine.

Preparation of buffer solutions.

  1. For each of the 2 buffer solutions prepare 10 labeled test tubes. Using the provided conjugate acid and conjugate base solutions mix them in the following ratios:

 

tube 1

tube 2

tube 3

tube 4

tube 5

tube 6

tube 7

tube 8

tube 9

tube 10

Volume of acid solution
(in ml.)

0.0

0.1

0.5

1.0

2.0

3.0

4.0

4.5

4.9

5.0

volume of base solution
(in ml)

5.0

4.9

4.5

4.0

3.0

2.0

1.0

0.5

0.1

0.0

 

For one set of reactions the acid form will be 0.05 M potassium dihydrogen phosphate. The base form of this set will be 0.05 M dipotassium hydrogen phosphate.

In the second set of reactions the conjugate acid will be 0.05 M Tris hydrochloride and the base will be 0.05 M Tris base.

2. Calibrate a pH meter and measure the pH of each of the solutions prepared on step 1.

3. Graph the data collected in step 2 with pH as the y-axis and log ([conjugate base]/[conjugate base]) as the x-axis. Note that you cannot use the tube 1 and tube 10 data. Why? This should be a linear function. Examine the Ka and pH definitions to convince yourself of this. Where can you find the value of pKa on this graph? How do your estimated pKa values compare with the literature values for these acid-base pairs?

4. Enzymatic Reaction: The enzyme will be prepared fresh at the start of each lab and should be stored on ice when not being used for a reaction. A peeled potato (2-3 cm cube) is ground in 50 ml of water for 1-2 minutes. The resulting homogenized solution is filtered through several layers of cheesecloth. The resulting filtrate is your crude enzyme solution. The enzyme solution should be kept cold and covered until used!

5. Prepare several reaction tubes. For each of the two buffer systems, choose ~3-4 pH values that span a fairly broad pH range. In each reaction tube, place 2 ml of your chosen buffer. To each reaction tube, then add 1 ml 0.01 M catechol (the substrate). Then add 1 ml of you crude enzyme solution, still keeping all material cool. Some important controls: you should prepare at least 1 tube with no enzyme but an equal amount of water. Similarly, you should prepare a tube with no substrate but an equivalent amount of water. Why are these controls important?

6. Transfer your reaction tubes into a 37 C water bath (or incubator). Since oxygen is a substrate, take each tube and agitate it every 5 minutes. At 5 minute intervals over 20-30 minutes, hold the tubes up to a light or white background and score them for any color change that occurs. (For example, you could use a - to +++ sort of system.) What can you conclude about the pH range in which this enzyme works best?

 

7. If your assay is successful, you should design a continuation experiment to further limit the conditions in which this enzyme works. Can you show it needs oxygen? What temperatures will it withstand? What temperatures will it work at? We will also try to bring in some known or suspected chemical inhibitors of this reaction.

7. Cleanup: Potato waste in garbage, any leftover enzyme or pH buffers may go down drain. Enzymatic reactions containing catechol and benzoquinone should be placed in the designated collection container.

Possible extensions. Take buffer solution vs. water and examine the effect on pH of adding dropwise acid/base (for example, 0.1 m HCl or NaOH)

 

Appendix: The Use of the pH meter.

The pH values are measured using an electronic pH meter. These meters contain at least two electrodes; the unit placed in solution contains both of these electrodes. One is a reference electrode. The second electrode is sensitive to hydrogen ion concentration. The most commonly used form of pH sensitive electrode is a glass electrode. This electrode allows the slow diffusion of cations through a slightly permeable glass surface. This slight permeability sets up an electrical potential that is dependent on hydrogen ion concentration. The meter then measures the electrical potential difference between the two electrodes and converts that into a pH value. It is difficult to measure absolute pH values more accurately than to within 0.1-0.3 pH units. However, these instruments can measure relative differences of pH between two solutions quite accurately-to within 0.001 pH unit; our instruments allow measurement to 0.01 pH unit. Before use, these instruments are calibrated using pH reference buffers of known pH. The lab staff will demonstrate this procedure during the lab.

A few other comments about the use of these pH meters-They work fairly well in the pH range of about 0.5-9. The electrodes are delicate and should not be allowed to dry out; there is an electrode storage solution provided. They are not very suitable for measuring the pH of very low concentration solutions. The relationship between voltage and pH, and the pH of the reference buffer both depend on temperature. There is a temperature compensation feature built into the meter.