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the fact that p-nitrophenylphosphate (pNPP) is a chromogenic substrate for alkaline phosphatase (and
most other phosphatases). This means that an aqueous solution of pNPP, which is colorless, turns
yellow when de-phosphorylated by alkaline phosphatase to form p-nitrophenol. This reaction is
summarized as follows:
p-nitrophenylphosphate (colorless) + H2O p-nitrophenol (yellow) + Pi (inorganic phosphate)
phosphatase Further, the degree of yellowing resulting from the accumulation of the product (p-nitrophenol) can be
measured with a spectrophotometer. A quantitative assessment of the activity of the enzyme is then
achievable with the use of Beer's law.
In this exercise we will conduct quantitative analyses of an enzyme out of its usual site of
action (in vitro) so that we can manipulate substrate concentration and pH. In more advanced courses,
you will learn how to extract and purify enzymes from biological material. Today, we will work with
purified alkaline phosphatase that was purchased from a commercial source. Biology 05LA – Fall Quarter 2012 Lab 5 – page 2 THE EXPERIMENTS:
Reaction Time – This trial is not a separate exercise. It is actually a part of each of the two exercises
that follow. The reason it appears separately is to give you some practice performing this kind of
experiment so that all of the remaining trials can be performed without error. Therefore, you should
repeat it as many times as necessary until you are comfortable with the results that you obtained. You
can then use the data collected here in each of the remaining exercises.
1. Prepare a sample tube with 5 ml of high pH buffer to use as your blank. Use this to zero the
spectrophotometer at 410 nm, and keep it for use at the beginning of each of the remaining
2. Prepare a second tube with 4.0 ml of substrate (stock concentration = 608 M).
3. Have a square of Parafilm ready. In the next step, you will want to add enzyme, top with
Parafilm, mix gently by inverting the tube three times (do not mix violently), and get the tube
into the spectrophotometer as rapidly as possible.
4. Add 1 ml of alkaline phosphatase and start timing immediately. (Make sure that you keep
your stock enzyme in an ice bucket!) 5. Take absorbance readings as soon as you can (marked 5, 7, 10 or whatever seconds), and at 30
seconds after you first added the enzyme. Then, take readings every 30 seconds, out to a total
of 10 minutes (if necessary). Remember that you can repeat this exercise if you think it is
necessary. 6. Graph absorbance versus time for the data you collected. ****
Substrate Concentration – In this exercise, we will examine the effect of varying substrate
concentrations on the rate of the reaction. The substrate concentrations we will use have been prepared
for you and are:
608 M (the stock dilution – already done)
0 M (all high pH buffer)
PLEASE NOTE THAT ALL OF THESE [SUBSTRATE]'S WILL GIVE A
DIFFERENT REACTION RATE.
1. Obtain 3 tubes and label each with one of the above substrate concentrations (remember, you
have already done the 608 M sample). Add 4 ml of the appropriate substrate concentration to
the proper tube and set aside.
2. Re-zero the spectrophotometer.
3. Beginning with the 304 M sample, proceed as described above in the reaction time trial.
4. Repeat this process for the remaining two samples.
5. Plot absorbance vs. time for each of the experimental trials on the same graph that you made
for the 608 M run. **** Biology 05LA – Fall Quarter 2012 Lab 5 – page 3 Effects of pH – Alkaline phosphatase, as the name implies, is most active at an alkaline pH. Examine
the effects of slightly acid pH by using the substrate pH 6.5 solution and the pH 6.5 enzyme.
1. Re-zero spectrophotometer.
2. Obtain 4.0 ml of "substrate pH 6.5" in a clean tube (concentration 608 M) – do not use the high
3. Have Parafilm square ready. Mix in 1.0 ml of low pH enzyme to the substrate tube, cap with
Parafilm, mix by inversion, and take absorbance readings every 30 seconds out to a total of 5
4. Plot absorbance vs. time for both the pH 6.5 and 10.4 (608 M) trials on the same axis. Remember
that the “Reaction time” trial was run at pH 10.4. Data Analysis:
Because you will be responsible for the analysis the data from both of the experiments, it is
important that you make sure that the raw data from both is recorded in your lab notebook prior to
leaving the lab.
Since the purpose of this laboratory is to evaluate the performance of alkaline phosphatase in a
variety of experimental conditions, some parameter related to the performance of the enzyme must be
used as a point of comparison for the different trials. We will use a value known as the “initial
velocity” of the reaction for this purpose. This value, abbreviated “v0”, represents the time segment of
the experiment when the reaction is proceeding at a constant and maximal rate for a given set of
experimental conditions. The units we will use for v0 will be mole...
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- Fall '12