An Investigation of Enzyme Activity:
Phosphatase
Objective:
Using the standard curve method to determine the activity of the enzyme phosphatase as
determined through spectrophotometric methods.
I. BACKGROUND MATERIAL
Figure 1: Phosphatase, like all other enzymes, is a
protein, complex and convoluted in its molecular
structure.
Enzymes are essential
for maintaining chemical
order in all cells (and life
in general); every cell
contains thousands of
different enzymes. In
this laboratory, you will
explore the way in which
enzymes work. The
particular enzyme you
will work with,
phosphatase, can
produce
p-nitrophenol, a colored
product. Thus, we can
measure how well the
enzyme is working
depending on how
intensely colored the
product solution is.
A. ENZYMES, GENERAL
Enzyme n: A complex protein produced by living cells that promotes a specific
biochemical reaction by acting as a catalyst.
Enzymes are catalysts, speeding up chemical reactions in cells, making reactions run to
completion thousands if not millions of times more quickly than if the enzyme was
absent. For example, carbonic anhydrase, which functions in red blood cells, is an
enzyme that converts 600,000 molecules of carbon dioxide to 600,000 molecules of
bicarbonate in ONE second. Other enzymes are not as efficient: Rubisco (ribulose 1,5
bisphosphate carboxylase /oxygenase), which functions in green plant leaves, fixes (adds)
3 molecules of carbon dioxide to 3 molecules of ribulose 1,5 bisphosphate in ONE
second.
The rate at which different enzymes convert substrate(s) to product(s) is highly variable,
and this rate is also subject to change due to environmental conditions, such as
temperature and the pH of the surrounding environment. For example, rates of
decomposition (a complex process whereby myriad chemical reactions occur, with
associated enzymes) are much slower in colder climates than in warmer climates;
decomposition is also slowed if not stopped all together in the highly acidic environment
of bogs.
An enzyme works on a substrate, which is then turned into a product (or products). In
one of the examples above, carbonic anhydrase is the enzyme, carbon dioxide is the
substrate it works on, and bicarbonate is the resulting product. In text, enzymes are easily
recognizable, as they usually end in the suffix –ase.
In this experiment, you will be measuring how quickly a product forms when an enzyme
is added to a substrate. The product formed will be colored, and the amount of product
produced can be measured in a spectrophotometer.
B. ENZYME ASSAY, GENERAL
(Assay n. Chemical testing carried out to determine the compositions of a substance or
the concentration of various components in a substance)
Enzyme assays measure how fast an enzyme works. The assays measure either the
decrease in substrate or the appearance of product. Often, enzyme assays use either
colored substrates or colored products so that concentrations of either can be measured in
a spectrophotometer. In a continuous assay, cuvettes with an enzyme reaction mixture
are placed in the spectrophotometer and a change in absorbance of a substrate or product
is measured over time (the reaction is occurring within the cuvette).
In the discontinuous enzyme assay, the reaction is stopped after a set period of time and
the absorbance is measured. Stopping the reaction requires a denaturing agent, such as
heat, or severe pH change, that deactivates the enzyme by changing its chemical
structure. All of the reacting mixture can be added to the denaturing agent at once, or
equal parts (or aliquots) of the reacting mixture can be removed from the reaction, put in
separate test tubes, and stopped individually, at different times since the beginning of the
reaction. The intensity of color in a given length of time is an indicator of the velocity
with which the enzyme has made product from substrate. This activity of the enzyme is
usually expressed in the units of moles/minute of product produced or substrate used.
C. PHOSPHATASE ASSAY
Phosphatases are enzymes that hydrolyze phosphate groups from a wide variety of
organic substrates (phosphate esters), producing an alcohol and phosphoric acid. They are
found in all cells and usually are classified as either acid phosphatases or alkaline
phosphatases. Phosphatases can be studied in crude cellular extracts or in pure form.
In this experiment an artificial substrate, p-nitrophenyl phosphate, will be used to study
the activity of a phosphatase enzyme. One of the products of the reaction, p-nitrophenol,
turns yellow under basic conditions and can be detected using a spectrophotometer. The
reaction is:
Substrate + Water + Enzyme  Product + Phosphoric acid + Enzyme
p-Nitrophenyl Phosphate + H2O + Phosphatase  p-Nitrophenol + H3PO4
+ Phosphatase
In the absence of the enzyme, the reaction would proceed very, very slowly.
In this lab you will monitor the increasing intensity of yellow color, indicating the
product, p-nitrophenol, is being produced. To quantify the amount of product, you will
first construct a standard curve that plots absorbance at 6 p-nitrophenol concentrations.
From this you can calculate the activity of phosphatase.
D. SERIAL DILUTION AND STANDARD CURVE
The idea behind the standard curve is simple. It is a common technique used to
determine the concentration of a substance, in which known concentrations of the
substance are first analyzed and graphed. The unknown concentration can then be
compared to these known concentrations.
Here, the standard curve you create will be based on the relationship between absorbance
and concentration. In this case you will be working with colored solutions. The first step
in creating a standard curve is to perform a series of serial dilutions. Take a hypothetical
colored solution with a concentration of 0.8 mg/mL. By removing half of this solution,
adding it to a new test tube, and adding an equal amount of distilled water (or colorless
buffer), the concentration of the second test tube will be 0.4 mg/mL. Next, by removing
half of this second concentration, adding it to a new test tube, and adding an equal
amount of distilled water, the concentration of this third test tube will be 0.2 mg/mL.
Repeat these steps again, and the concentration in a hypothetical fourth test tube will be
0.1 mg/mL, and so on. Eventually, we will end up with multiple test tubes, each with half
the concentration of the former.
Figure 2: Test tubes from
serial dilution and unknown
sample. The clear test tube is
used to “blank the spec.”
0.8 mg/ml 0.4 mg/ml 0.2 mg/ml 0.1 mg/ml 0 mg/ml (Blank)
unknown…..CONCENTRATION
0.9
0.34
0.45
0.22
0.11
…….ABSORBANCE
Graphing these known concentrations against their absorbance results in a straight line.
(Remember that A = Ecl.) This linear graph is confusingly called the standard curve.
Now, the sample of unknown concentration can also be measured for absorbance, and its
concentration can be solved.
Figure 3: A standard curve for the test tubes shown above. The
concentration of the unknown substance has been solved.
II. A CONTEXT FOR THE EXERCISE
Recall the steps in Scientific Process:
1. Make observations about the natural world.
2. Ask questions about, or formulate a reasonable testable hypothesis to explain
observations.
3. Design and execute, experiments to generate results, which could answer the question
or test the hypothesis.
4. Analyze results and draw inferences regarding the veracity of the hypothesis and, if
true, how it might fit with “established” knowledge.
You should keep those steps in mind as you approach the analysis of any laboratory
exercise.
Observations: As indicated in the text, most chemical reactions occur at a rate, which is
too slow to support standard life functions. Thus a catalyst, called an enzyme, is
required.
Question: How does the presence of the enzyme affect the rate of the chemical reaction?
Hypothesis: Knowing that “Phosphatase” removes a phosphate group from an organic
compound, you might propose something similar to:
The rate of the conversion of p-nitrophenol phosphate to p-nitrophenol will be
increased if the enzyme phosphatase is present.
Experiment: The procedure you will employ is a standard enzyme assay, which is
outlined below. In order to assess that amount of product produced by the reaction
you will create a standard curve for the concentration of p-nitrophenol and use
spectrophotometry to test your hypothesis. See Methods, section III.
Results: Record your results in self-explanatory tables and graphs. Do your results differ
from/agree with your classmates? What inferences can you make from your own
results or from compiled results? Do they correspond with the results expected
form the previous literature?
III. METHODS
A. Standard Curve Determination
1.
Using serial dilution techniques, prepare 1 mL each of six p-nitrophenol
(PRODUCT) concentrations, ranging from 15.6 to 500uM. Use the stock pnitrophenol solution and the 0.05 M Standard Buffer to make the dilutions in 6
marked test tubes. Hint, you start with 2 mL, in the first tube, but end with 2 mL
in the last tube, and only have 1 mL remaining in the first 5 tubes.
2.
Take 0.5 mL of each dilution you made in step 1, and add it to 4.5 mL of 0.02 M
NaOH. (This is done because the structure of p-nitrophenol is such that it only
appears yellow in the presence of a base.) What are the concentrations of these
new solutions? Why do you need to know?
3.
Make sure the solutions are well mixed (vortex them briefly).
4.
Set the wavelength of the spectrophotometer to 410 nm and blank (set the
absorbance to zero) with a cuvette of 0.02M NaOH.
5.
Transfer each dilution (prepared in #2) to a cuvette (enough to fill) and measure
the absorbance of each at 410 nm.
6.
Graph your absorbance data against the concentration of the solutions measured,
and draw a line of best fit through the points. The resulting best-fit line is your
standard curve. Why do you fit a line to these data points, and not connect the
dots?
B. Standard Phosphatase Reaction Assay
You are performing a discontinuous enzyme assay. The reaction occurs for a total of 10
minutes, but an aliquot of the reaction mixture is removed every two minutes and stopped
(for a total of 5 aliquots). In the end, you will have a test tube with a 2-minute-old
reaction, one with a 4-minute-old reaction, one with a 6-minute-old reaction, and so on.
Each of these test tubes can be assessed individually in the spectrophotometer. You
must also consider what control reaction is important to test your hypothesis and set
it up also. The directions below are for the experimental treatments. You must
determine how to set up the control.
1.
Prepare 5 “stop test tubes” of 4.5 mL of 0.02 M NaOH. This strong base will halt
the enzyme reaction by denaturing the enzyme, and turn your product yellow.
You will stop the reaction 5 times.
2.
Start the reaction. The reaction mixture is simple, made of three parts:
a. 3 mL of 0.05 M Standard (sodium citrate) Buffer, pH 4.8
b. 3 mL of 1mM p-nitrophenyl phosphate (substrate)
c. 3 mL of 1 mg/mL phosphatase
The buffer and substrate are added to a clean test tube. Then, the enzyme is added
to initiate the reaction, the tube is quickly and briefly stirred (vortex), and the
stopwatch is started. Remember that the reaction starts immediately after the
enzyme is added, so work quickly!
3.
The reaction is run for a total of 10 minutes, BUT at successive 2 minute intervals
during the 10 minute-long reaction, pipette 0.5 mL of the mixture from the
“reaction tube” into one of the 5 “stop tubes” containing 4.5 mL of 0.02 M NaOH.
4.
Three minutes after adding an aliquot of the reaction to a “stop tube,” fill a
cuvette with this second mixture. Measure the stopped mixture’s absorbance in a
spectrophotometer at 410 nm. (Make sure to “blank the spec” first with NaOH)
5.
At the end of the ten-minute reaction, you should have a total of 5 test tubes of
reaction stopped at different intervals (2, 4, 6, 8 and 10 minutes) and their
associated absorbances.
6.
Using the Standard Curve that you generated, calculate the concentration of pnitrophenol at each time period. Graph the results with time on the x-axis and [pnitrophenol] on the y-axis.
IV. QUESTIONS
1. Compare your results to other lab groups. Do your individual results support or fail to
support your hypothesis? Do the compiled results support or fail to support the
hypotheses?
2. Is the standard curve you
created for the product, pnitrophenol, linear? Why do
you think it is or is not?
3. How much p-nitrophenol
was produced after two
minutes of running the
reaction? After four minutes?
How about six minutes? Is the
relationship linear? Why or
why not? What does the shape
of the line say about the
reaction?
4. It is often convenient to give
an “averaged activity” rate for
a given enzyme. Did the
enzyme’s activity rate change
over time, and if so, is it
appropriate to assign an
averaged activity rate for phosphatase in this reaction? If you had to choose an averaged
activity rate for phosphatase, which point in the reaction would be most indicative of the
overall activity over 10 minutes?
5. If we are to simply express enzyme activity rate as a measure of velocity, what other
units could we use besides moles of product produced per minute?
6. Does the activity of an enzyme depend on the amount of substrate or enzyme present?