Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
EXTRACTION OF ALKALINE PHOSPHATASE FROM E-COLI AND ITS
PURIFICATION USING DIFFERENT METHODS
VICTOIRE NDONG
LAB PARTNER: ELINA SHRESTHA
BIOCHEMISTRY 311: TUESDAY LAB SECTION
INSTRUCTOR: LAURIE LENTZ-MARINO
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
ABSTRACT
Alkaline phosphate is a hydrolaze enzyme that can be found between the cell wall and the
cell membrane of the E-coli bacterium. It is responsible for taking off a phosphate group from
many molecules that play important role in many metabolism reactions, a process called
dephosphorilation. It is very resistant and its optimum pH is around 10.
The purpose of our five weeks experiment was to extract alkaline phosphatase from Ecoli bacteria and to purify it week after week using different method such as column
chromatography, dialysis, heat inactivation and ammonium precipitation. To determine the
purity of the enzyme we checked the protein levels every week and we also made enzyme assays
to see how the activity of the enzyme was progressing. For every week we checked that the
enzyme activity increases and the protein level decreases.
RESULTS
Over the past five weeks, we have worked on isolating an enzyme and purifying it with
different methods every week. For every stage of the enzyme we got, we checked the purity of
the enzyme by measuring its enzyme activity and its protein level. For the first week, the enzyme
was isolated from the E coli and the extract was placed in a dialysis bag to allow small molecules
to leave the bag and to retain the big molecules. Our results for the first week are shown on Table
1 and figure 1 (attached).
For the second week, the dialysis product was heated to denature the proteins and then it
was precipitated using Ammonium sulfate in order to precipitate the proteins and remove some
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
remaining lipids. Once again the sample was tested for enzyme activity and the rest of the stage
was put into another dialysis tube this time to remove the excess ammonium sulfate. The results
for week two are show on table 2 and figure 2 (attached).
For the third week, we used our stage 3 retentate from the dialysis tube and we run it
through a DEAE-Sephacel gel like structure that binds proteins containing anionic groups. At the
end we collected the purified enzyme in 20 different tubes and we did an enzyme assay of the 20
tubes and we checked for the tube with the highest absorbance to be our stage 4. Unfortunately
our results we got were not very accurate so for day 5 we used another group’s stage 4. We also
measured the activity of stage 3 on graph 3 (attached)
Table 3: Fraction from the column chromatography and their absorbance value from the
spectrophotometer
Fraction Absorbance (A410)
Activity detected
number
1
-0.0698
2
-0.0597
3
-0.0619
4
-0.0703
5
-0.0569
6
-0.0711
7
-0.0386
8
-0.0694
9
-0.0642
10
-0.0698
11
-0.0453
12
-0.0321
13
-0.0444
14
0.0018
15
-0.0226
16
-0.0509
17
-0.0499
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
18
19
20
0.0208 No
activity(Fraction
18)
-0.0285
-0.1954
For the fourth week, we used the four different stages we got so far plus the alkaline
phosphatase and the low and high protein molecular weight standards to compare the amount of
protein on each and also to see if the amount of Alkaline phosphatase increased with each stage.
We loaded lane 1 to 7 of our gel and we kept it for one week. On week 5 we took a picture of our
gel we run the previous week. We also did an enzyme activity assay to compare the enzyme
activity of samples with different concentrations (.25, .125, .0625, .03, and .015) with and
without inhibitor. The results were recorded using the spectrophotometer. Our results were not
correct for that reason we used Laurie’s values for our plot. We plotted the values both on a
Michaelis Menton plot and a Lineweaver-urk plot to learn more about the enzymes
characteristics.
Table 4: Results of the enzyme activity assay of the enzyme with and without inhibitor (our
results)
Concentration reaction without
reaction + 0.1 mM
Reaction + 0.3
in mM
inhibitor
inhibitor
mM inhibitor
0.25 mM
1.7214
1.1016
0.8879
0.125 mM
1.0806
0.4173
0.5902
0.0625 mM
0.5882
0.2983
0.03 mM
0.2827
0.2792
0.1822
0.015 mM
0.1446
0.147
0.0677
Laurie’s Data
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
[S] (mM)
0.012
0.024
0.05
0.1
0.2
Rate
Rate
0.0528
0.0801 0.006
0.1019 0.009
0.1201 0.023
0.127 0.06
Rate
0.0044
0.011
0.016
0.0337
1/[S] (mM- 1/v
1/v
1/v
1)
(min)
(min)w/Pi (min)
w/o Pi 0.1mM
0.3mM
83.33
18.93
41.67
12.48
169.49
227
20
9.81
112.36
90.9
10
8.33
43.1
62.5
5
7.85
17.09
30
Figure 4: Plot of the rate of appearance of the product versus the substrate concentration with and
without inhibitor
Figure 5: Line weaver-burk plot of the inverse of the velocity vs the inverse of the concentration
of the enzyme (with and without inhibitor)
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
Calculations:
1/V0 =1/Vmax + Km/Vmax (1/[s])
Vmax for graph 1 (inhibitor 0.3 mM) is 1/ 6.941 = 1.001
Vmax graph 2 (inhibitor 0.1 mM) 1/6.3458 = 0.1576
Vmax graph 3 (without inhibitor) 1/0.999 = 0.144
Km is the substrate concentration at which the rate is half Vmax. The slope is Km/Vmax
Km (without inhibitor)= 0.020, Km(0.1 mM) = 0.6509 Km(0.3 mM) = 5.306
Calculation of α and Ki
At 0.1 α = Km’/Km =0.6509/0.020= 28.3
α = 1+[I] / Ki
Ki = [inhibitor]/ [α-1] = 0.1/27.3 = 0.0037
For the 0.3mM inhibitor
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
α = Km’/Km = 5.306/0.020= 265.30
Ki = 0.3/ 264.30 = 0.001. The average Ki = (0.0037 + 0.001) / 2 = 0.002
Table 5: Proteins present on our gel and their Rf values
Protein
Molecular
weight
Log (m.w.)
Rf
Myosin
200000
5.3
0.23
B-galactosidase
116250
5.07
0.3
Phosphorylase b
97400
4.99
0.33
Serum albumin
66200
4.82
0.4
Ovalbumin
45000
4.65
0.52
Carbonic
Anhydrase
31000
4.49
0.6
Trypsin inhibitor
21500
4.33
0.67
APase
?
?
0.47
Figure 6: Plot of the protein molecular weights vs the Rf values
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
The logarythm of the Apase molecular weight is Alkaline phosphate is 4.74 so it’s molecular
weight is 54954.08 and the molecular weight of each subunit is 27477.04.
Table 6: Data for overall progression of the purification of the enzyme
Fractio
n
Volume
of
Fraction
(ml)
Stage 1
8.7
Stage 2
mU/mL
Total
mg
proteins
in
fraction
Specific Fold
yield
Activity purification
(mU/mg)
4945.5 43025.85 12.23
106.4
404.38
1
100%
11.9
1726.1 20540.59 1.26
14.99
1370.29
3.39
47.74%
Stage 3
2.1
416.25
0.4
2185.31
5.40
2.03%
Stage 4
3.5
-
-
-
-
-
Total
units in
Fraction
(mU)
mg/mL
874.125 0.20
-
-
DISCUSSION:
The purpose of our experiment was to isolate E-coli alkaline phosphatase which is found
between the cell wall and the cell membrane of the E-coli. We purified the enzyme week after
week using different methods like dialysis, heat denaturation and column chromatography and
we measured its purity by checking its activity and also its protein level. The purer the enzyme is,
the higher its activity, and the lower its protein level.
If we look at the overall progression of the enzyme, we can see that its activity has
increased over the weeks but also its yield was very different from one experiment to another.
The most efficient method to purify the enzyme is supposed to be the one that gives the higher
activity and a good yield. The first enzyme assay we performed showed a curve with a slope of
0.3297 and the protein concentration was 12.23 mg/ml. Stage 1 was dialyzed to purify the
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
enzyme and separate it from other molecules. In dialysis, the cellulose bag is semi permeable
permitting small molecules to move out and still keeping the big molecule. The activity of the
enzyme was low compared to the other weeks’ results and also the protein level in stage one was
higher than the other stages because it wasn’t as pure. The product of the dialysis of stage 1 was
called stage 2.
On the second week we got a big yield 136% of stage 2 because the dialysis caused small
molecules to move out and big molecules to stay in. The fact that many of our tubes were placed
in the same container may also have had an effect on the exchange of molecules making our
yield bigger more than 100%; water molecules moved from one tube to another depending on the
osmotic pressure. The result of the enzyme assay showed us much more activity of the enzyme:
the slope was 0.4603 and the protein concentration was 1.26 mg/ml. Stage 2 was heat denatured
to make the proteins that are still contained in the enzyme insoluble thus separate them more
from the enzyme. The alkaline phosphate that is contained in the substance was not affected by
the heat because it was still stable at that temperature. After heat denaturating, we used
ammonium sulfate which is a salt at different concentrations precipitates proteins of different
solubility. That method permits to remove some proteins but it mainly allows the concentration
of the enzyme. This method is called “differential precipitation”. The product was put again in
the dialysis tube but this time, it was to remove the ammonium sulfate used for the precipitation.
The product of that dialysis was stage 3.
On the third week, we measured the enzyme kinetics of stage 3 and surprisingly, this time
the slope was 0.1110 which is smaller than the value we got for stage 1. However, the protein
level was way lower than for the two first stages it was 0.20 which made us assume that the
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
result we got was due to a mistake in the preparation of the enzyme assay or maybe the big
decrease in yield (17%). Stage 3 was used for the DEAE-Sephacel column chromatography
which is made up of cellulose. The DEAE-cellulose binds to proteins which contain negatively
charged groups. It’s important the pH is above the pI of the proteins because the isoelectric pH is
when all the sum of all charges is 0. Above the pI, the pH is basic and the net charge of the
proteins will be negative allowing them to be displaced by the gel. If the ammonium sulfate was
not removed by dialysis, it would hinder the chromatography process because it is a neutral
molecule and it wouldn’t have been stopped by the gel. The column fractions that were collected
in 20 were used to assay the enzyme activity so that the one with the highest absorbance will be
used as the Stage 4 fraction. Unfortunately, we didn’t get a good absorbance for any of our tubes
and we had to use other groups’ stage 4.
On week four, we took all stages: 1-4 along with low and high molecular weight protein
standards and alkaline phosphatase and we run them in a gel. The goal of gel electrophoresis is to
separate different proteins depending on their size. The proteins with the lowest molecular
weight will run faster in the gel and the biggest protein will be more at the top. Running our
stages with different standards helped us see what different type of proteins was present in our
stages and also their sizes. The alkaline phosphatase also helped localize the enzyme and see
how clear the band was on each stage. When we looked at our gel, the first stage looked like a
smear with a lot of different bands. There were many proteins present plus the alkaline
phosphatase. That is because the first stage was not very pure and there were still a lot of
enzymes left from in the fraction. Stage 2 looked clearer that stage 1 and it had a band at the
alkaline phosphatase stage. However there were still some proteins left. Their Rf values almost
corresponded to proteins like ovalbumine, the trypsin inhibitor and serum albumin. Stage 3
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
looked very clear and there was no band at the alkaline phosphatase site. That might explain why
we didn’t get any stage 4 after the column chromatography. Our enzyme might have been left in
the DEAE column. The band for alkaline phosphatase got fainter from one stage to another that
might be because of the loss in yield and in total enzyme.
On the fifth week we made a lot of enzyme assays to check the behavior of the alkaline
phosphatase in the presence of inhibitors at different concentration. The inhibitor in our
experiment was Pi (inactive phosphate). To help us study the inhibitor we used the Lineweaver
burk plot and the M.M plot. The values of Vmax we got for the enzyme at different
concentrations were not very different: 0.1576 for the 0.1mM Pi and 0.144 for reaction without
inhibitor. The Km values however were considerably different: 0.6509 for the 0.1 mM inhibitor
and 0.020 for the regular reaction. The inhibitor doesn’t affect the Vmax but it increases the Km;
that makes us believe that Pi is a competitive inhibitor that binds to the active site of the enzyme
and competes with the substrate. The Ki of the enzyme is 0.0025. The activity of our alkaline
phosphatase was 2185.31mU/mg and the turn over of E-coli was 480 thus the Mr of our alkaline
phosphatase was 86000. The purity of our stage 3 is (S.A x 8.6/4800) = 3.92%.
During our five weeks enzyme has gotten purer and purer however a lot of factors such as
the protein levels or the method used had an effect on the results. Some methods are very
efficient in purifying the enzyme but they are not very good at giving good yield. It is important
during the extraction and purification of the alkaline phosphatase that the method gives both a
good purification of the enzyme and also a good yield. Stage 3 was the clearest of all our stages
but the yield was very small making the method of heart purification and ammonium
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Victoire Ndong
Lab partner: Elina Shrestha
Biochemistry311: Tuesday lab
precipitation less efficient than we thought. But in general we reached our goal of purifying the
enzyme week after week.
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