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 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)
number
Activity detected
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
18
19
0.0208 No activity(Fraction
18)
-0.0285
20
-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
Table 4: Results of the enzyme activity assay of the enzyme with and without inhibitor (our results)
Concentration reaction without
in mM
inhibitor
0.25 mM
0.125 mM
0.0625 mM
0.03 mM
0.015 mM
reaction + 0.1 mM
inhibitor
1.7214
1.0806
0.5882
0.2827
0.1446
1.1016
0.4173
0.2792
0.147
Reaction + 0.3 mM
inhibitor
0.8879
0.5902
0.2983
0.1822
0.0677
Laurie’s Data
[S] (mM)
Rate
Rate
Rate
0.012 0.0528
1/[S] (mM1)
1/v
1/v
1/v (min)
(min)
(min)w/Pi 0.3mM
w/o Pi
0.1mM
83.33
18.93
0.024 0.0801 0.006
0.0044
41.67
12.48
169.49
227
0.05 0.1019 0.009
0.011
20
9.81
112.36
90.9
0.1 0.1201 0.023
0.016
10
8.33
43.1
62.5
0.0337
5
7.85
17.09
30
0.2
0.127
0.06
Figure 4: Line weaver-burk plot of the inverse of the velocity vs the inverse of the concentration of the
enzyme (with and without inhibitor)
Lineweaver-Burk plot
500
400
1/velocity (min)
300
1/v (min) w/o Pi
200
1/v (min)w/Pi 0.1mM
1/v (min)w/Pi 0.3mM
100
-100
-70
-40
0
-10
y = 5.3003x + 0.999 [0.3mM Pi]
20
-100
50
80
y = 4.1304x + 6.3458 [0.1mM Pi]
y = 0.1419x + 6.9413
-200
-300
1/[pNPP] (mM-1)
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(3)= 0.020, Km(2) = 0.6509 Km(1) = 5.306
Calculation of α and Ki
At 0.1 α = Km’/Km =0.6509/0.020= 28.3
Ki= [inhibitor]/ [α-1] = 0.1/27.3 = 0.0037
For the 0.3mM inhibitor
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: Protein 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
Day 1
We did the enzyme isolation, enzyme assay, and dialysis.
Fraction
Volume
ml
enzyme assay
Total units in
fraction
mg/ml nanodrop
µg/µl=mg/ml
Yield
Stage 1
8.7
4945.5
43025.85
12.23
Stage 2
11.9
1726.1
20540.59
1.26
136%
0.2
17.6%
Stage 3
2.1
Stage 4
3.5
Fraction
Rate dA/min
Stage 1
0.3297
Stage 2
0.4603
416.25
874.125
0
166%
Stage 3
0.111
Day 2
We did the Heat denaturation and ammonium sulfate precipitation and enzyme assay.
Day 3
Column chromatography and enzyme assay we searched for stage four out of all the samples we had.
Day 4
We did gel electrophoresis to examine the protein composition of the fractions, and an enzyme assay
for that we didn’t get a stage 4 we also did the nanodrop.
Day 5
We summarized the whole thing and we looked at the gels took pictures of it and we measured the
distance between the bands.
Concentration reaction without
in mM
inhibitor
0.25 mM
0.125 mM
0.0625 mM
0.03 mM
0.015 mM
reaction + 0.1 mM
inhibitor
1.7214
1.0806
0.5882
0.2827
0.1446
1.1016
0.4173
0.2792
0.147
Reaction + 0.3 mM
inhibitor
0.8879
0.5902
0.2983
0.1822
0.0677
The purpose of our experiment was to isolate E-coli alakaline 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.
The first step of the five week experiment was to isolate the enzyme by breaking down the cell wall of
the bacteria using lysozyme which hydrolyzes parts of the cell wall and we also added EDTA which
removes Ca++ from the cell and DNase that hydrolizes the DNA. After isolating the enzyme (which we
called stage 1) we did an enzyme assay and checked the kinetics of the substance. The first results we
got showed us.
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 enzyme and separate it from other molecules. In dialysis,
the cellulose bag is semi permeable permiting 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 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.