Isolation, Quantification, Protein Gel Electrophoresis, &

Enzyme Kinetics of Alkaline Phosphatase

Melvin Onyia, Maria Hernandez, Eric Ovalle, TK Lee, & Daniella Jayanty
Dr. Neha Parikh
CHEM 4140: CRN 11053
December 12, 2014
Words: 1640

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Abstract
The purpose of the experiment was to observe different qualities of Alkaline
Phosphatase using biochemistry laboratory techniques such as chromatographic isolation,
spectrophotometricquantification,electrophoresis, andenzymekineticsanalysis.
These principal techniques demonstrate the procedures required to remove AP from the
E.coli bacterium and isolate it for analyze its activity. The resulting data indicates there is
greater enzymatic activity and grater affinity at pH 7.0. Alkaline Phosphatase can be used
for various scientific endeavors that play key roles in groundbreaking scientific research
due to its stability.

Introduction
The initial step is to isolate E. coli Alkaline Phosphatase (AP) using dialysis and
column chromatography. E. coli Alkaline Phosphatase is located in the periplasm, it is a
heat-stable homodimeric enzyme with a molecular weight of 86,000 and contains two
Zn2+ per dimer with a pI of 4.5 and a maximal activity at pH 8.0. An E. coli K-12 mutant
bacterium was prepared for the experiment.
Spectrophotometryservestoquantifyanestimateofthecompoundsbasedon
colorintensities.Theuseofcolorimetricproceduresisusefultomeasurethe
concentrationofproteins,determinationofenzymatickineticconstants,andmeasuring
ligandbindingreactions.Thisexperimentquantifiedtheproteinconcentrationofalkaline
phosphataseusingtheBradfordproteinassayprocedureforabsorbanceat595nm.
Electrophoresis is a versatile tool in analytical biochemistry; it
allows the separation of proteins and other molecules based on a

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varying number of properties. The mobility of ions is determined by the

charge (q) of the molecule, the voltage gradient of the electric field (E),
and the fractional resistance of the supporting medium (f). The ratio of
the distance a protein migrates and a small anionic dye is referred to
as the relative motilities of said proteins (Rf). Ammonium persulfate
and TEMED are used to form free radicals which in turn react with
acrylamide and induce polymerization.
The use of steady state kinetics to determine the mechanism of
an enzyme can be quite useful. Characteristics of kinetic properties
include determining the Km, Vmax, and inhibition constant of various
substances. The velocity of catalyzed reaction can be determine by the
rate limiting step k2. Michaelis constant Km represents the
concentration of substrate that produces half of the maximum velocity
of the catalyzed reaction. Km allows for the determination of the
amount of substrate needed to reach the maximum velocity and
indirectly signifies enzyme affinity.

Materials
E. coli K-12 was suspended at 25 mg/ml in 10 ml of 30 mM Tris-HCl (pH 8.0),
0.5 M Sucrose. 5 g of E. coli was washed with 10 mM Tris-HCl and centrifuged and resuspended in 10 ml of 30 mM Tris-HCl (pH 8.0),0.5 M Sucrose. Lysozyme, DNAse, and
MgSO4, EDTA, and Tris-HCl were used to isolate the enzyme.High-speed centrifuge
was used initially at 12,000g at 4 C (rpm = 91(g): 9969 rpm); N606. Ammonium

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sulfate, (DEAE), and Sigma-Fast BCIP/Nitro-blue Terazonium Tablets were used to test
the final concentration of AP
Aspectrophotometerwassetto595nm.TherequiredamountofBSAstandard
(0.2mg/mL)withColumnbufferA(Dilutant):5mMTrisHCL,5mMMgCl2,pH7.4
andsamples(S1,S2,S3,S4)werepipettedintotheappropriatetesttubesofthe96well
plate.0.2mLofworkingBradfordreagentwasaddedtoeachtesttubeandmixed.The
totalvolumeofproteinsampleineachtubewas50L,absorbancewasmeasuredviathe
platereader.
A stacking gel was prepared with 50 L of 25% ammonium
persulfate, 10L of TEMED, and 7 mL of 10% acrylamide gel mix. 20 L
of each stage (1-4) were mixed with 20 L of 2X SDS-PAGE and boiledto
denatureproteins, used as sample buffer, stored at 4 C. A protein
standard ladder, AP stages 1-4 and pure alkaline phosphatase were
pipetted into separate wells. The gel was placed into a Bio-Rad MiniPROTEAN 3 Cell gel kit with 1 liter of 1X SDS-PAGE and went through
electrophoresis at 200 volts. 1 liter of coomassie blue stain was used,
after which 2 rounds of 1 liter of destain solution was used.
3 mL of 0.2 M Tris_HCl and 3 mL 50 M p-nitrophenol (PNPMW=139.1
g/mole

) were placed into a reference cuvette. 1.5 mL of 1 mM p-

nitrophenyl phosphate (PNPPMW=457.4 g/mole) and 1.5 ml of Tris-HCl (pH 8.0)

were pipetted into a secondary reference cuvette; to be used to zero
the spectrometer. 2.5 mL of 0.2 M Tris-HCl (pH 8.0) and 0.45 ml of 1
mM PNPP were placed into a cuvette. 50 L of enzyme was added and

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mixed quickly. The absorbance was measured in 20 sec. increments for

180sec. The change in absorbance was measures every 20 seconds
with 0.25, 0.5, 1, 10, 25, 50, 100 and 150, 200, 300, 400, 500 and 600
M PNPP in the presence of 50 l enzyme. All absorbance were
measured at 410 nm.

Results
1. Initially 19 mL of the solution was collected, after several days in the dialysis
buffer, the solution expanded to 43 mL. Volume after centrifuge was 41 mL. The final
dialysis and centrifuge resulted in a volume of 13 mL. Figure 1-1 & 1-2 show the results
of the test and the efficiency of separating AP for other proteins. The violet color in
Figure 1-3 indicates the concentration alkaline phosphatase. The volume of the collected
portion of the mixture was 9.5 mL.
2.Figure21graphsthestandardBSAcurve.Thecorrelationvalueof0.985
showedthattheabsorptiontoconcentrationratiowaswithinrangeof100%.Thefinal
concentrationofAPisdisplayedintheFigure22.ThemeasuredtotaloftheStage1
enzymewas876.12mg/ml.Theresultsofeachofthesesstageswere118.79mg/mland
228.42mg/mlrespectively.ThemeasuredtotaloftheStage4enzymewas9.77mg/ml.
3. Figure 3-1 displays the destained electrophoresis gel. Stage 1
displays 8 bands in the third well. The Stage 2 enzyme displays 3
bands in the fourth well. The stage 3 enzyme displays 2 bands in the
fifth well, and the stage 4 enzyme displays 2 bands in the sixth well.
The commercial AP is blown out at 86 kDa in the seventh well.

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4. Figures 4-1 and 4-2 display the data and corresponding graph
of the absorbance of AP. Figures 4-3, 4-4, 4-9, & 4-10 display the data
regarding the pooled samples of AP and their concentrations at pH 8.0
and 7.0. Figures 4-5, 4-6, 4-11, & 4-12 display the data comparing Vo
to substrate concentration at pH 8.0 and 7.0. Figures 4-7, 4-8, 4-13, &
4-14 display data comparing 1/Vo and 1/[S] at pH 8.0 and 7.0. The
Vmax and Km at pH 8.0 are 44.44 and 8.88. The Vmax and Km at pH
7.0 are 103.09 and 3.44.

Discussion
The resulting concentration of AP did not follow the ideal curve due to some level
of impurity. Inconsistency could be due to left over ammonium phosphate from previous
steps.TheassayBSAvalueGhadthemostnegativeeffectontheR2valueandwas
omitted.Thefourthstagealkalinephosphataseresultedat9.7mg/mL.Thisamountis
smallcomparedtothestartingamountofproteinanalyzedintheinitialstages.
Figure 3-1 shows that the Stages 2-4 were not as visible as stage
one. This is due to the fact that there are more proteins present in the
sample during the initial stage of AP isolation. The band where AP are
expected, progressively become lighter from Stages 1-4, this is due to
the increased purity and isolation of alkaline phosphatase. Stage one
contains 8 bands; each with a clear and visible band, showing that
there is a high amount of the proteins. The subsequent bands become
almost indistinguishable, and the pure AP shows a clear large band,

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expressing that there is a high concentration of pure AP as expected.

The experiment could be improved by using a larger sample size as
well as a stain with higher sensitivity such as silver. Another alternative
is to use a stronger camera, which may improve the visibility of the
stains.
The absorbance of PNP on the stage 1-4 enzymes displayed in
Figures 4-1 and 4-2 shows there is a greater level of absorption during
the second stage compared to the other stages. At this stage there are
more compounds present in the solution. This shows there is positive
enzymatic activity. Figure 4-6 displays a standard logarithmic trend
line, which demonstrates an expected substrate to enzyme kinetic
activity. This is not the case in Figure 4-12, which is at pH 7.0. The
trend line has displayed an inverted logarithmic curve and the
scattered points do not correlate to a positive enzymatic activity. The
Lineweaver-Burk plot at pH 8.0 displays a more concentrated
arrangement that is more expected than the inconsistent plot at pH
7.0.
The absorption of PNPP in the pooled data at pH 7.0, shows some
level of discrepancy. The data appears skewed and inconstant with the
data collected for pH 8.0. Although the enzymatic activity is expected
to be higher at pH 8.0 versus pH 7.0 this still does not explain the
statistical discrepancies. The data for pH 7.0 had an affect on the
subsequent data that relied on the initial measurements. The Vmax and

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Km at pH 8.0 are 44.44 and 8.88. The Vmax and Km at pH 7.0 are
103.09 and 3.44. This shows that at pH 7.0 there is a greater
efficiency of the enzyme.
The stated pH for maximal enzymatic activity was 8.0. As you
can see in Figure 4-4 compared to Figure 4-7, the concentration of
PNPP is dramatically higher. This shows there is greater affinity of the
enzyme at pH 7.0. The data at pH 7.0 does give an ideal representation
of the kinetic activity and catalytic activity of alkaline phosphatases;
this is duetodifferentpoolsofenzymestage4usedinthetwoexperiments.

Appendix

AP Enzyme Stages
2500
2000
1500
mL 1000
500
0

Holiday Period New Year's Day Memorial Day

Procedure

AP Enzyme Stages

Figure 1-1

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Alkaline Phoshatase Concentration

1
0.8
0.6
Concentration 0.4
0.2
0
Test Tube Fractions

Figure 1-2

Figure 1-3

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BSA Standard Curve

1.5
1

Absorbance (595 nm) 0.5

f(x) = 0.01x + 0.04

R = 0.99

0
0 50 100150200250

BSA Concentration (mg/ml)

Figure 2-1

Average Protein Concentration

Concentration mg/mL

1000
500
0

AP Stages

Figure 2-2

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Figure 3-1
Absorbance of PNP for Stage #1-4 Enzymes

Stage 1
0.228

0.298
0.295
0.29
0.286
0.281
0.279
0.277
0.276
0.275
0.274

Stage 2
Stage 3
Stage 4
0.228
0.25
0.227
0.278
0.247
0.402
0.287
0.256
0.486
0.316
0.278
0.59
0.351
0.3
0.659
0.383
0.321
0.739
0.415
0.344
0.846
0.443
0.368
0.947
0.474
0.393
1.056
0.502
0.417
1.141
0.532
0.438

Figure 4-1. Table displaying the absorbance of PNP for Stage # 1-4
Enzymes

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Absorbance of PNP for Stage #1-4 Enzymes

1.2
1

Stage 1

0.8
Absorbance (nm)

Stage 2

0.6

Stage 3

0.4

Stage 4

0.2
0
0

50

100

150

200

Time (s)

Figure 4-2. Graph displaying the absorbance of PNP for stage #1-4
enzymes.

Concentration of PNP [M] at pH 8.0

Time (secs)
Abs 0.0 mM
NPP
Abs 0.01 mM PNPP
Ab
0.025 mM
NPP
Abs 0.05
mM PNPP
Abs
0.1 mM PNPP
Abs
0.15 mM PNPP
Abs 0.2 mM PNPP
Abs 0.3 mM P
PP
Abs 0
4 mM PNPP
Abs 0.5 mM PNPP
Abs 0.6 mM PNPP
5
47.25897
21
283
553875
378.0718336

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45.1795841
1181.47448
614.3667297
661.6257089
992.4385633
708.8846881
992.4385633
1181.47448
20
0
803.4026465
1086.956522
1323.251418
1323.251418
1039.697543
1417.769376
1748.582231
1937.618147
1512.287335
1937.618147
40
47.25897921
1323.251418
1512.287335
1843.100189
2032.136106
1512.287335
2835.538752
2599.243856
3260.869565
2173.913043
2410.20794
60
0
1937.618147
2457.466919
2835.538752
2930.056711
2032.136106
3969.754253
3544.42344
4584.120983
2882.797732
3260.869565
80
47.25897921
2410.20794
3166.351607
3544.42344

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3827.977316
2551.984877
5056.710775
4773.1569
6001.890359
3497.164461
4064.272212
100
0
2882.797732
3827.977316
4253.308129
4631.379962
3071.833648
6096.408318
6001.890359
7419.659735
3875.236295
4820.415879
120
47.25897921
3166.351607
4442.344045
5009.451796
5529.300567
3733.459357
7230.623819
7136.10586
8837.429112
4300.567108
5529.300567
140
0
3591.68242
5056.710775
5718.336484
6379.962193
4300.567108
8364.839319
8317.58034
10349.71645
5151.228733
6285.444234
160
47.25897921
3969.754253
5623.818526
6427.221172
7183.364839
4584.120983

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9404.536862
9404.536862
11814.7448
5907.372401
7041.587902
180
47.25897921
4206.049149
6049.149338
7088.846881
7939.508507
5482.041588
10491.49338
10491.49338
13279.77316
6663.516068
7844.990548

Figure 4-3. Table displaying concentration of PNP [M] at pH 8.0

Concentration of PNP vs Time (s) at pH 8.0

14000.000

Abs 0.0 mM PNPP

12000.000

Abs 0.01 mM PNPP

10000.000

Concentration of PNP (M)

Abs 0.025 mM PNPP

Abs 0.05 mM PNPP

8000.000

Abs 0.1 mM PNPP

6000.000

Abs 0.15 mM PNPP

4000.000
2000.000
0.000

Abs 0.2 mM PNPP

Abs 0.3 mM PNPP
Abs 0.4 mM PNPP
Abs 0.5 mM PNPP
Abs 0.6 mM PNPP

Time [sec]

Figure 4-4. Graph plotting concentration of PNP [M] at pH 8.0.

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Vo vs. Substrate at pH 8.0

S [mM]
S [M]
Vo
0
0
0
0.01
10
22.41425871
0.025
25
32.40615717
0.05
50
35.10667027
0.1
100
38.61733729
0.15
150
27.8152849
0.2
200
56.17067243
0.3
300
54.28031326
0.4
400
71.83364839
0.5
500
32.40615717
0.6
600
38.07723467

Figure 4-5. Table displaying Vo vs. Substrate at pH 8.0

Vo vs. Substrate at pH 8.0

80
60
Vo

40
20
0
0

100

200

300

400

500

600

700

S[M]

Figure 4-6. Graph plotting Vo vs. Substrate at pH 8.0 with logarithmic

trend line.

1/s (mM)
100
40
20
10

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1/s (M)
0.1
0.04
0.02
0.01

1/Vo
0.044614458
0.030858333
0.028484615
0.025895105

6.666666667
5
3.333333333
2.5
2
1.666666667

0.006666667
0.005
0.003333333
0.0025
0.002
0.001666667

0.035951456
0.017802885
0.018422886
0.013921053
0.030858333
0.026262411

Figure 4-7. Table displaying Linweaver-Burk data.

Lineweaver-Burk Plot at pH 8.0

0.05
0.04
0.03
0.02

1/Vo

0.01
-0.2

-0.15

-0.1

0
-0.05
0
-0.01

0.05

0.1

0.15

-0.02
1/s[M]

Figure 4-8. Graph of Linweaver-Burk plot at pH 8.0 with trend line.

Concentration of PNP [M] at pH 7.0

Time (secs)

0.0 mM PNPP
0.01 mM PNPP
0.025 mM PNPP
0.05 mM PNPP
0.1 mM
P
0.15 mM PNPP
0.2 mM
PNPP

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0.3 mM PNPP
0.4
M PNPP
0.5 mM PNPP
0.6 mM PNPP
5.000
0.000
785.340
523.560
8115.183
7329.843
8115.183
1047.120
785.340
785.340
1832.461
1308.901
20.000
0.000
1832.461
1570.681
13350.785
15183.246
17801.047
2356.021
1570.681
1570.681
2879.581
2094.241
40.000
261.780
3141.361
2879.581
21204.188
29057.592
29319.372
3664.921
2879.581
3141.361
4188.482
4188.482
60.000
0.000
4188.482
3926.702
28272.251
40575.916
40314.136
4188.482
4188.482

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4712.042
6020.942
5497.382
80.000
0.000
5497.382
5235.602
35078.534
51570.681
50785.340
5235.602
5759.162
6020.942
7853.403
6806.283
100.000
0.000
7591.623
7329.843
41361.257
61780.105
62041.885
6544.503
7329.843
7329.843
8900.524
8376.963
120.000
0.000
8900.524
8638.743
47120.419
71204.188
69371.728
7591.623
8900.524
8900.524
10471.204
9947.644
140.000
0.000
10209.424
9947.644
52879.581
79842.932
78795.812
8900.524
10732.984
10471.204
12041.885

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11518.325
160.000
0.000
11518.325
11256.545
57853.403
88481.675
87696.335
10209.424
12303.665
11518.325
13612.565
13089.005
180.000
0.000
11780.105
12565.445
62827.225
96858.639
96073.298
11256.545
13612.565
12827.225
15183.246
14921.466

Figure 4-9. Table displaying concentration of PNP [M] at pH 7.0.

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Concentration of PNP vs Time (s) at pH 7.0

120000
100000
80000

Concentration of PNP (M)

Abs 0.0 mM PNPP

Abs 0.01 mM PNPP
Abs 0.025 mM PNPP
Abs 0.05 mM PNPP

60000

Abs 0.1 mM PNPP

40000

Abs 0.15 mM PNPP

20000

Abs 0.2 mM PNPP

Abs 0.3 mM PNPP

Abs 0.4 mM PNPP
Abs 0.5 mM PNPP
Abs 0.6 mM PNPP

Time [sec]

Figure 4-10. Graph plotting concentration of PNP [M] at pH 7.0.

Vo vs. Substrate at pH 8.0

S [mM]
S [M]
Vo
0
0
0
0.01
10
62.82722513
0.025
25
68.81077038
0.05
50
312.6402393
0.1
100
511.5931189
0.15
150
502.617801
0.2
200
58.33956619
0.3
300
73.29842932
0.4
400
68.81077038
0.5
500
76.29020194
0.6
600
77.78608826

Figure 4-11. Table displaying Vo vs. Substrate at pH 7.0

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Vo vs. Substrate at pH 7.0

600
500
400
Vo

300
200
100
0
0

100

200

300

400

500

600

700

S[M]

Figure 4-12. Graph plotting Vo vs. Substrate at pH 8.0 with logarithmic

trend line.

1/s (mM)
100
40
20
10
6.666666667
5
3.333333333
2.5
2
1.666666667

1/s (M)
0.1
0.04
0.02
0.01
0.006666667
0.005
0.003333333
0.0025
0.002
0.001666667

1/Vo
0.015916667
0.014532609
0.003198565
0.001954678
0.001989583
0.017141026
0.013642857
0.014532609
0.013107843
0.012855769

Figure 4-13. Table displaying Linweaver-Burk data.

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Lineweaver-Burk Plot at pH 7.0

0.02
0.02
0.01
0.01
1/Vo

0
-0.2

-0.15

-0.1

-0.05
0
-0.01

0.05

0.1

0.15

-0.01
-0.02
-0.02
1/s[M]

Figure 4-14. Graph of Linweaver-Burk plot at pH 8.0 with trend line.

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