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Cite this: DOI: 10.1039/x0xx00000x Specific Activity of Acid Phosphate and Effects of pH and
pKa on PNP
Halle Spanke (BIO 541L Section 002) (TA: Cailin McCracken)
Received 00th January 2012,
Accepted 00th January 2012
The main purpose of this experiment was to determine the absorbance maxima and pKa of
DOI: 10.1039/x0xx00000x para-nitrophenol (PNP), the Km and Vmax of Acid Phosphatase, as well as the specific
activity of Acid Phosphatase. This was done through three separate experiments over the span
www.uwec.edu/ of three weeks. During the first experiment the absorbance maxima was found by making a a
sample cuvette with 0.5mL of 0.1 mM PNP mixed with a 1.5 mL buffer pH 10 and a blank
cuvette with 2.0 mL of a pH 10 buffer and placing both in the spectrophotometer. Eight other
cuvettes were prepared with varying pH values and the wavelength of maximal absorbance for
PNP was determined from 360nm – 430 nm in 10 nm increments using the spectrophotometer.
In the second experiment 6 cuvettes with equal amounts of 0.1 M Tris pH 7.2 and 0.1 M
MgCl2 and differing amount of H20 and substrate 50mM pNPP were made. 6 separate trials
were done with the enzyme 5 mg/mL Phosphatase to determine the absorbance recorded over
a period. This data was used to determine the initial velocity which then helped create a
Lineweaver-Burk equation to determine the Km and Vmax of the phosphatase enzyme. During
the third experiment 5 test tubes were set up with equal amount of diH20, 0.1 M Tris pH 7.2,
and 0.1 M MgCl2 and differing amounts of acid phosphatase. Once the test tubes were set up
0.5 mM pNPP was added to each test tube and incubated for 10 minutes. After 10 minutes 5 M
NaOH was added to each test tube to stop the reaction and the solutions were placed in
cuvettes where the absorbance was recorded from the spectrophotometer. The product PNP
present and the concentration were determined using the Beer-Lambert Law which then
allowed the specific activity of the enzyme phosphatase to be calculated. The results that were
obtained include the following; absorbance maxima = 400 nm, pKa of PNP = 7.2, Km= 2.228
mM, Vmax=0.0051921 mM/s, specific activities = 113.67 U/mg, 349.021 U/mg, 209.306
U/mg, and 305.9375 U/mg. This data is important for understanding enzymes and substrate
interactions

Introduction Hasselbach equation, pH=pKa+log([A-]/[HA]).
The pKa indicates how strong or weak an acid can
Three separate experiments were conducted to be. Lower pKa values represent a strong acid and a
determine the absorbance maxima and pKa of PNP, higher pKa value represents a weak acid. The
Km and Vmax values of Acid Phosphatase, and buffering capacity of acid/conjugate base is greatest
specific activities of Acid Phosphatase. A buffer is when pH=pKa. The Beer-Lambert Law can be used
a solution that can resist pH change with the to calculate specific activity which was needed
addition of acidic or basic components. This is during the third experiment conducted. Beer-
important when conducting reactions that need a Lambert Law is demonstrated by A=ebc where
specific and stable pH range since pH represents either A(absorbance) is being calculated or c
the H+ ions and the buffers will help reduce any (concentration of a substance). The two other
major changes in H+ concentration. The pH of a variables e (extinction coefficient) and b (cell
buffer depends on the pKa of the acid and the ratio pathlength) are usually known variables.
of acid and its conjugate base concentrations. This Spectrophotometry is a quantitative measurement
can be demonstrated using the Henderson –

This journal is © The Royal Society of Chemistry 2013 J. Name., 2013, 00, 1-3 | 1
ARTICLE Journal Name
technique that allows investigation for optical mixing 0.5 mL of 0.1 mM PNP and 1.5 mL
properties of materials over a wide wavelength buffer 10. 2 mL of each of the remaining 8 test
range [2]. This was used in all three experiments tubes was transferred into cuvettes. All the
cuvettes were then brought to the
when measuring the different absorbance levels in spectrophotometer. To set up the
each reaction. Enzymes can generally lower the spectrophotometer the wavelength was adjusted
activation energy by reducing the energy needed to. 360 nm and B-cuvette was placed into
for reactants to come together and react. Since position 1 and the S-cuvette was placed into
enzymes bring together reactants so they do not position 4, the spectrophotometer was then
have to expand energy moving around until they zeroed. The S-cuvette was moved into a position
come together randomly. Enzymes bind to a for the absorbance to be read and it was
recorded. This was repeated for the wavelength
substrate at a specific site called the active site and 360 nm through 430 nm in increments on 10 nm.
by doing this they do not have to overcome Between each wavelength the
intermolecular forces [5]. This way the molecules spectrophotometer was blanked, and each
interact with less energy. The Michaelis-Menten absorbance was recorded. The absorbance
equation can be expressed as maxima was 400 nm. The spectrophotometer
v=(Vmax[S])/(Km+[S]) and is used to characterize was then set to 400 nm and blanked. The 8
cuvettes with various pH levels were then placed
enzyme kinetics. The Vmax in the equation helps into the machine four at a time and the
represent the maximum velocity achieved by the absorbance was recorded.
system at maximum substrate concentration. Km For the second experiment 6 cuvettes were
then represents the substrate concentration at which labelled. #1-6. The following reagents were
the reaction velocity is half of the Vmax [4]. In the pipetted into different cuvettes: 0.1 M Tris pH
second experiment the Michaelis-Menten equation 7.2, 0.1 M MgCl2, H20,and 50 mM pNPP. The
helped describe the function of the enzyme table below demonstrates how much of each
reagent was added:
function in relation to the concentration of the
substrate that was present [4]. The specific activity
of an enzyme can help define the purity of that
enzyme within the protein mixture. The unit of the
specific activity is important as it is important in
the purification of enzymes from proteins [3]. Acid
phosphatases are a group of enzymes that can be
found in many animal and plant species. The
function of acid phosphatase is to remove The spectrophotometer was set to 400 nm and
phosphate from other molecules during digestion. reaction 1 was placed in the machine and zeroed.
This enzyme is the most effective in acidic The enzyme 5mg/mL phosphatase was added to
environment but was used in the experiments reaction 1 and the absorbance was recorded
where the pH levels varied. immediately. The absorbance was then recorded
every 20 seconds for the span of 120 seconds.
This process was repeated for the 5 other
Experimental reactions and the spectrophotometer was
For the first experiment pH strips were used on blanked between each reaction.
each of the 8 different buffer solutions to For the third experiment 5 test tubes were set up
determine the corresponding pH levels. The 8 with the following reagents: diH20, 0.1 M Tris
test tubes were then labelled with various pH pH 7.2, 0.1
levels (5,6,7,7.5,8,8.5,9,10). 1.0 mL of 0.1 mM M MgCl2, Acid Phosphatase. The table below
PNP were added to each tube using a pipettor demonstrates how much of each reagent was
then an automatic pipettor was used to add 3.0 added to each test tube.
mL of each buffer to its corresponding test tube.
There were 10 total cuvettes used in this
experiment. One with a B (blank), one with a S
(sample), and 8 with the various pH levels. To
create a buffer, 2.0 mL of pH 10 was added to
the B-cuvette and the S-cuvette was created by

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In each test tube 300 microliters of 0.5 mM pnPP

was added and each were incubated at room
temperature for 10 minutes. After the 10 minutes
each reaction was stopped by adding 2mL of 5
M NaOH to each of the test tubes. 1000
microliters of each reaction were transferred to
the corresponding cuvette. Reaction 5 was used
to blank the spectrophotometer since no
enzymes were added then the other 4 reactions
were placed and in and their absorbance was Table 3: S, Vi, 1/Vi, 1/s, 1/v (EXP 2)
recorded at 400 nm.
Results

Table 1: Wavelength and absorbance of PNP (EXP

1). Figure 3: Lineweaver Burk Plot (EXP 2)
Absorbance maxima of PNP = 400 nm
Vmax: 0.0051921 mM/s
Km: 2.228 mM

Figure 1: pH vs. Absorbance of PNP (EXP 1) Table 4:Absorbance of Reactions 1-5 at 400nm
pKa= 7.2 (EXP3)
Beer Lambert Law Calculations (EXP 3):
RXN 1: 0.055/18000 = 3.06 x 10 ^-6 M x
1000mM=0.0031mM
RXN 2: 0.275/18000 = 1.53 x 10 ^-5 M x
1000mM= 0.01523 mM
RXN 3: 0.246/18000 = 1.37 x 10^-5 M x 1000
Table 2: Absorbance of PNP at 400 nm for mM=0.0137 mM
Reactions 1-6 (EXP 2) RXN 4: 0.481/18000 = 2.67 x 10^-5 M x
1000mM=0.0267 mM
RXN 5: 0 mM
Specific Activity Calculations (EXP 3):
RXN1:0.0031mmol/Lxmin(1L/10^3mL)(10^6n
mol/1mmol) =3.1nmol/mLxmin
3.1nmol/mLxmin(2.75mL)=8.525U
(.5)(.15)=.075mg
8.525U/.075mg = 113.67 U/mg
RXN2:0.01523mmol/Lxmin(1L/10^3mL)(10^6
Figure 2: Time(s) vs. Absorbance for Reactions nmol/1mmol) =15.23nmol/mLxmin
1-6 (EXP 2) 15.23nmol/mLxmin(2.75mL)=349.021U

This journal is © The Royal Society of Chemistry 2012 J. Name., 2012, 00, 1-3 | 3
ARTICLE Journal Name
(.8)(.15)=.12mg of acid phosphatase that was put in for this
41.8825U/.12mg = 349.021 U/mg reaction. Reaction three also could have had
some type of error as it was lower than reaction
RXN3:0.0137mmol/Lxmin(1L/10^3mL)(10^6n 2 even though there was more acid phosphatase
mol/1mmol) =13.7nmol/mLxmin used. This error could be that the correct amount
13.7nmol/mLxmin(2.75mL)=37.625U of acid phosphatase was not used for this
(1.2)(.15)=.18mg reaction causing it to be lower than reaction 2.
37.625 U/.18mg = 209.306U/mg These values were then used to calculate the
specific activity for each of the reactions which
RXN4:0.0267mmol/Lxmin(1L/10^3mL)(10^6n were calculated to be: 113.67U/mg,
mol/1mmol) =26.7nmol/mLxmin 349.021U/mg, 209.306U/mg, 305.9375U/mg
26.7nmol/mLxmin(2.75mL)=73.425U for reactions 1-4. Again reaction 5 was the
(1.6)(.15)=.24mg control and had a specific activity of 0U/mg. For
73.425U/.24mg = 305.9375 U/mg these values there was no accurate trend that was
determined. This could again be from errors
RXN5: 0 U/mg during the experiment which could include the
wrong about of acid phosphatase to be added
Conclusions when using the pipettor, wrong amounts of the
other reagents to be used, or error when using the
Through each of the three experiments spectrophotometer such as placing the cuvettes
conducted accurate measurements of absolute in wrong or not blanking it correctly. The
maxima and pKa of PNP, Km and Vmax of acid specific activity represents the ratio of enzyme
phosphatase, and specific activity of acid activity to enzyme concentration which then
phosphatase were found. In. the first experiment helps determine the enzyme purity in the
400 nm was concluded to be the absolute solution. There could be a few actions taken to
maxima since .294 was the highest value. The prevent error if this experiment were to be
pKa was determined to be 7.2 which was close repeated. The first being having whoever is
to the expected value of 7.15 [1]. Figure 1 was using the equipment such as the pipettor and the
used to determine the pKa from looking at the spectrophotometer having more practice with
slope. The data that was determined from this lab those tools. Being able to know how to properly
helps see how acidic the solutions were based on use these tools could have prevented error within
the pH. For the second experiment the Km was the reagents amount used for all the experiment
calculated to be 2.228 mM which represents the and gaining correct absorbance values. Having
substrate concentration at which the reaction more background on what was happening during
velocity is half of the Vmax. The Vmax for this these experiments also could have prevented
experiment was calculated to be 0.0051921 error. By having a better understanding of the
mM/s. Both values were solved through using experiment’s errors could have been found while
the Figure 3 which was the Lineweaver Burk doing the experiments and if there was a need to
Plot. The y-intercept represents 1/Vmax, the x go back and redo some of the reactions after
intercept represents -1/Km, and the gradient noticing inaccurate trends then error in data
represents Km/Vmax [4]. These values can vary could have been prevented. For the third
based on the enzyme’s affinity to its substrate. experiment enzyme was almost completely
For example, an enzyme with a high Km value frozen which could have caused issues in
has a low affinity for its substrate [4]. Based off gathering the correct amount. Making sure all
Figure 2 it can be concluded that each of the the materials are ready to go before conducting
reactions had different absorbances. This can the experiment could have prevented an error
most likely be attributed to the varying amounts like this one. This experiment could be taken
of H20 and 50 mM pnPP for each reaction. For further by using lower pH levels during the first
the third experiment the product concentration experiment and seeing how the data collected
were calculated using the Beer-Lambert law and from that compares to the data from this
the calculated values were 0.0031mM, experiment.
0.01523mM, 0.0137mM, 0.0267mM for the References
reactions 1-4 and reaction 5 was a control
reaction. The main outlier in these calculations
would be the value from the first reaction as it
had a much lower value than the others. This
could be from human error or from the amount

4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 2012
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