An Investigation of Enzyme Activity: Phosphatase
Objective:
Analyze how the activity of the enzyme phosphatase is influenced by factors in its environment.
Laboratory Overview:
Most enzymes are proteins that are essential for
maintaining chemical order in all cells; every cell
contains thousands of different enzymes. In this
laboratory, you will explore how the environment
influences enzyme function. 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.
Enzyme Background:
Enzymes function to promote a specific biochemical
reaction by acting as a catalyst. Catalysts speed up
chemical reactions in cells, making reactions run to
Figure 1: Phosphatase, like all other
completion thousands if not millions of times more
enzymes, is a protein, complex and
quickly than if the enzyme was absent. For example,
convoluted in its molecular structure.
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.
With enzymes chemical reactions happen at speeds that are compatible with life. There is variation in
the rate at which an enzyme speeds up a chemical reaction. This rate is also subject to change due to
environmental conditions, such as temperature and pH of the surrounding environment. For example,
rates of decomposition (breakdown) 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). For example, the
enzyme carbonic anhydrase uses carbon dioxide as the substrate and produces bicarbonate as the
product. In this experiment, you will measure the amount of product formed when an enzyme is added
to a substrate. The product formed will be colored and the amount can be easily measured using a
spectrophotometer (spec).
Enzyme Assay:
Enzyme assays (experiments) determine how fast an enzyme works by measuring either the decrease
in substrate or the appearance of product. These assays are performed in test tubes or similar container
and often use colored substrates or products for quick measurement of concentration in a
spectrophotometer. The activity of the enzyme can be monitored continuously in a spec by taking
measurements at given intervals. Conversely, a portion of the assay is removed, treated to stop enzyme
activity and then measured; this is a discontinuous measure of activity and the one you will use in lab.
The change in color over a given length of time indicates the rate that the enzyme worked to make
product from substrate. This rate is usually expressed in the units of µmoles/minute of product
produced, or substrate used.
In Figure 2 you see an example of data collected from an
enzyme assay. The amount of product formed has increased
over time. Initially, the enzyme works as fast as it can to
produce product. Over time the enzyme will have less and
less substrate available so it will form less and less product.
Eventually the enzyme runs out of substrate and no more
product is formed. For the laboratory today we are most
interested in how quickly the enzyme is working in the
beginning, called the initial rate. This part of the curve is
assumed to be linear and the slope is equal to the initial
rate. In the lab, the initial rate of phosphatase activity will
be compared under different environmental conditions to
see how it changes.
Figure 2: A graph
illustrating the plot of data
collected from an enzyme
assay.
Phosphatase:
In lab you will work with an enzyme called a phosphatase. These enzymes eliminate phosphate groups
from a wide variety of substrates; they are found in all cells and are usually classified as either acid
phosphatases or alkaline phosphatases.
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
You will monitor the increasing intensity of yellow color using a spec; this color indicates the product,
p-nitrophenol, is being produced. To quantify the amount of product and phosphatase activity you will
utilize a standard curve that shows the relationship between p-nitrophenol concentration and
absorbance, the unit of measure in a spec. The standard curve is a common technique used to
determine the concentration of a substance in a sample. Known concentrations of the substance are
first analyzed and graphed to establish the relationship between quantity and absorbance; the
concentration of a substance in an unknown sample is then determined using this curve.
Spectrophotometer and Absorbance:
A spectrophotometer is an instrument used to measure the relative intensities of light wavelengths in a
spectrum. Visible light is composed of colors with distinct wavelengths. Spectrophotometry is based
on the principle that substances absorb light in particular wavelengths and reflect light in other
wavelengths. Each substance has an “absorption signature,” measured as the amount of absorption for
each wavelength. The amount of
light absorbed is dependent upon
the concentration of the substance
(e.g. p-nitrophenol) in solution.
A spectrophotometer works by
projecting a beam of light of a
particular wavelength through a
solution contained in a cuvette, a
test tube made of optically clear
Figure 3: Diagram illustrating the basic structure of a
glass or plastic (Figure 3). The
spectrophotometer.
solution will absorb some of the
light and the amount absorbed is
proportional to quantity of molecules in the solution that take up the light. This proportionality is
called the Beer-Lambert Law and is expressed by the following equation:
A = Elc
This relationship states that the absorption (A) of light of a particular wavelength is proportional to the
length of the light path through the solution (l) times the concentration (c) of the substance. The E in
the equation is the molar extinction coefficient that is a constant for a particular wave length, and
particular substance. Different substances absorb different amounts of a particular wavelength of light
and therefore have different E values. Note that there is a linear relationship between absorbance and
concentration. As the concentration of the absorbing substance increases, the amount of light it
absorbs increases.
Standard Curve:
As mentioned previously, the standard curve in this lab is based on the relationship between
absorbance and concentration. A series of increasingly dilute samples of p-nitrophenol, with known
concentrations, are made (Figure 4) and the absorbance of each one is determined. These data are
graphed and the relationship yields a straight line (Figure 5). Once the absorbance of an unknown
sample is determined this standard curve is used to determine the concentration of p-nitrophenol
(product) in an enzyme assay.
Figure 4: Test tubes from serial
dilutions and an unknown sample.
The clear test tube is used “blank”
the spectrophotometer.
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
Figure 5: This is the standard curve you will use
to calculate the concentration of p-nitrophenol in
all your enzyme reactions during the lab.
Preparation for the Experiment:
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 pH or temperature affect enzyme function?
Hypothesis: An example might be:
As temperature increases, phosphatase activity increases.
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 use a standard curve for the
concentration of p-nitrophenol and use spectrophotometry to test your hypothesis.
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 support or refute your hypotheses?
Experimental methods:
Spectrophotometer instruction summary:
The “spec” is a very delicate instrument. When using a spec, please be careful and ask your TA if you
encounter any difficulties! As with any delicate machine, using it well takes practice.
1) Allow spec to warm up for 30 minutes before use.
2) Press the “A/T/C” button to change the spec between modes; absorbance, transmittance, and
concentration. You want to measure absorbance, so make sure the display indicates absorbance mode
(A).
3) Press the “nm ∆” or “nm ∇” button to select the wavelength you want to measure.
4) Insert a cuvette filled with the “blank” solution into cell holder, and close sample door. The blank
accounts for any absorbance that would occur simply from the solvent (dissolving liquid).
5) Press the “0 ABS/100% T” to zero the absorbance for the “blank” solution.
6) Remove blank and insert a cuvette filled with your sample into cell holder. Close the door and read
the absorbance value on the display.
Phosphatase Assay:
You are performing a discontinuous enzyme assay. The reaction is run for a total of 10 minutes, but an
aliquot (small portion) of the reaction mixture is removed every two minutes and added to a solution
that stops enzyme activity. In the end, you will have a sample 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 samples can be
assessed individually in the spectrophotometer. The directions below are for the experimental
treatments.
Half of the groups in the lab will conduct experiments to investigate the affect pH has on enzyme
activity, and half will conduct experiments to investigate the affect temperature has on enzyme
activity. Follow the procedure for the experiments your group will test.
Temperature experiments:
For these experiments, you will utilize water baths to run the enzyme assays at three different
temperatures; 4°C, room temperature, and 65°C. Read through the entire procedure before you do
anything to be sure you understand what you will do!
1.
For each temperature to be tested, prepare 5 “stop” cuvettes with 1.8 mL of 0.02 M NaOH
solution in each. Use a pipettor with a clean tip, but you do not need change tips between
cuvettes since the solution is the same. This strong base will stop the enzyme reaction, and turn
the enzyme product yellow so it can be measured in the spectrophotometer.
2.
You will need to prepare 3 sets (two tubes) of reaction solutions in the test tubes provided, one
set of 2 for each temperature to be tested. The reaction mixture is made of three parts; buffer,
enzyme substrate, and enzyme. Be careful to identify the solutions correctly before you use
them!
To begin, using a pipettor with a clean tip for each different solution, add to a clean test tube:
a. 1.0 mL of 0.05 M citrate buffer, pH 5
b. 1.0 mL of 1mM p-nitrophenyl phosphate (substrate) in buffer at pH 5
To a second clean test tube (with a clean tip!) add:
c. 1.0 mL of 1 mg/mL phosphatase in buffer at pH 5
Place both tubes into the appropriate temperature water bath for 5 minutes to stabilize at the
correct reaction temperature. For the room temperature experiment, simply leave the test tube
on the counter in the rack. Swirl the tubes periodically to help them come to temperature.
3.
When you are ready to run the reaction, pour the buffer/substrate solution into the tube
containing enzyme to initiate the reaction, quickly cap (gloved finger) and invert the tube
several times to mix, start the stopwatch, and return the tube to the water bath. Remember that
the reaction starts immediately after the enzyme is added, so work quickly!
4.
At each 2 minute interval from the start, pipette 0.2 mL (200 µL) of the mixture from the
reaction into one of the 5 “stop” cuvettes containing 1.8 mL of 0.02 M NaOH. You can use the
same pipette tip for each aliquot from the reaction.
5.
After the reaction is complete, and all 5 cuvettes are filled, use a clean gloved finger to cover
the cuvette and invert it several times to mix thoroughly. Dry the glove with paper towel
between cuvettes. Wait an additional 2 minutes after mixing, then measure the absorbance of
each cuvette at 410 nm. Make sure to “blank the spec” first with 0.02 M NaOH.
pH experiments
For these experiments, you will run the enzyme assays at three different pH levels; 3, 5, and 7. Read
through the entire procedure before you do anything to be sure you understand what you will do!
Be careful to identify the solutions correctly before you use them!
1.
For each pH to be tested, prepare 5 “stop” cuvettes with 1.8 mL of 0.02 M NaOH solution in
each. Use a pipettor with a clean tip, but you do not need change tips between cuvettes since
the solutions are all the same. This strong base will stop the enzyme reaction, and turn the
enzyme product yellow so it can be measured in the spectrophotometer.
2.
You will need to prepare 3 reaction solutions in the test tubes provided, one for each pH to be
tested. The reaction mixture is made of three parts; buffer, enzyme substrate, and enzyme.
Each reaction must use buffer, enzyme, and substrate at the same pH. For these experiments,
you have pH 3, pH 5, and pH 7 solutions to work with. Each of the three solutions you need
are provided all three pHs, so be careful what you use!
To begin, using a pipettor with a clean tip for each different solution, add to a clean test tube:
a. 1.0 mL of appropriate pH buffer
b. 1.0 mL of 1mM p-nitrophenyl phosphate (substrate) at the appropriate pH
Swirl the tubes to mix them.
3.
When you are ready to run the reaction, add 1.0 mL of 1 mg/mL phosphatase at the correct
pH (with a clean tip!) to the tube containing the buffer/substrate solution to initiate the reaction,
quickly cap (gloved finger) and invert the tube several times to mix, start the stopwatch, and
return the tube to the rack. Remember that the reaction starts immediately after the enzyme is
added, so work quickly!
4.
At each 2 minute interval from the start, pipette 0.2 mL (200 µL) of the mixture from the
reaction into one of the 5 “stop” cuvettes containing 1.8 mL of 0.02 M NaOH. You can use the
same pipette tip for each aliquot from the reaction.
5.
After the reaction is complete, and all 5 cuvettes are filled, use a clean gloved finger to cover
the cuvette and invert it several times to mix thoroughly. Dry the glove with paper towel
between cuvettes. Wait an additional 2 minutes after mixing, then measure the absorbance of
each cuvette at 410 nm. (Make sure to “blank the spec” first with 0.02 M NaOH)
Data analysis
Now that your assays are complete, you will have 5 absorbance values for each of your three
experimental treatments. These absorbance readings must be manipulated in order for them to be
useful in answering the original questions that our experiments were intended to address.
1.
Use the standard curve provided to determine the concentration of enzyme product (pnitrophenol) at each assay time period. Graph these values with time on the x-axis, and the
concentration of p-nitrophenol on the y-axis. Fit a curve to the points as best you can, noting
the sample curve provided (Figure 2). You should plot all three reactions on the same graph.
2.
On your graph, identify the region of each curve which best represents the initial rate of the
reaction (see Figure 2), before the curve begins to flatten, if at all. The slope, steepness, or
gradient of this line (represented as m) relates to how fast the enzyme was able to convert
substrate to product. The faster the enzyme worked (higher initial rate), the more product that
was created in any time period.
From geometry, we know that the slope of a line is:
So, the slope of the line on your graph can be thought of as the change in product concentration
(x) divided by the change in time (y):
The faster the enzyme worked, the more product created per time period, the higher slope
(larger m) the reaction curve will have.
Observe your graph, and compare the initial reaction rates of the three reactions.
Discussion Questions:
1) Compare the initial reaction rate of the reactions conducted at each different temperature or pH.
Summarize your results. Are the results consistent between groups?
2) Write down your conclusion(s) based on the results. Do the conclusions support your
hypothesis?
3) One of the features of life is the maintenance of homeostasis. Homeostasis is the ability of an
organism to preserve constant internal conditions despite fluctuations in the external
environment. Given what you have observed about the effects of pH and temperature on
enzyme function, explain why maintenance of homeostasis is crucial to life.
References:
1) Spectrophotometer image:
http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Reaction_Rates/Experimental_Deter
mination_of_Kinetcs/Spectrophotometry
2) Enzyme assay image:
http://en.wikipedia.org/wiki/Enzyme_kinetics