Announcement: Bring
your biology lab notebooks to the afternoon session on Monday October 16.
Goals:
Useful
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:
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 + ½ 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.
|
|
tube 1 |
tube 2 |
tube 3 |
tube 4 |
tube 5 |
tube 6 |
tube 7 |
tube 8 |
tube 9 |
tube 10 |
|
Volume of acid solution |
0.0 |
0.1 |
0.5 |
1.0 |
2.0 |
3.0 |
4.0 |
4.5 |
4.9 |
5.0 |
|
volume of base solution |
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.