1

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,

spectrophotometric quantification, electrophoresis, and enzyme kinetics analysis. 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.

Spectrophotometry serves to quantify an estimate of the compounds based on

color intensities. The use of colorimetric procedures is useful to measure the

concentration of proteins, determination of enzymatic kinetic constants, and measuring

ligand-binding reactions. This experiment quantified the protein concentration of alkaline

phosphatase using the Bradford protein assay procedurefor absorbance at 595 nm.

Electrophoresis is a versatile tool in analytical biochemistry; it allows the

separation of proteins and other molecules based on a varying number of

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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 re-

suspended 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 = 91Ö(g): 9969 rpm); N606. Ammonium

sulfate, (DEAE), and Sigma-Fast BCIP/Nitro-blue Terazonium Tablets were used to test

the final concentration of AP

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A spectrophotometer was set to 595nm. The required amount of BSA standard

(0.2 mg/mL) with Column buffer A (Dilutant): 5 mM Tris-HCL, 5 mM MgCl2, pH 7.4

and samples (S1, S2, S3, S4) were pipetted into the appropriate test tubes of the 96 well

plate. 0.2 mL of working Bradford reagent was added to each test tube and mixed. The

total volume of protein sample in each tube was 50 µL, absorbance was measured via the

plate reader.

A stacking gel was prepared with 50 µL of 25% ammonium persulfate, 10µL

of TEMED, and 7 mL of 10% acrylamide gel mix. 20 µL of each stage (1-4) were

mixed with 20 µL of 2X SDS-PAGE and boiled to denature proteins, 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

Mini-PROTEAN 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 µM 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 µL of enzyme was

added and 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 µM PNPP in the presence of 50

µl enzyme. All absorbance were measured at 410 nm.

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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. Figure 2-1 graphs the standard BSA curve. The correlation value of 0.985

showed that the absorption to concentration ratio was within range of 100%. The final

concentration of AP is displayed in the Figure 2-2. The measured total of the Stage 1

enzyme was 876.12 mg/ml. The results of each of theses stages were 118.79 mg/ml and

228.42 mg/ml respectively. The measured total of the Stage 4 enzyme was 9.77 mg/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.

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

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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. The assay BSA value “G” had the most negative effect on the R2 value and was

omitted. The fourth stage alkaline phosphatase resulted at 9.7 mg/mL. This amount is

small compared to the starting amount of protein analyzed in the initial stages.

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, 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

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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

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 due to different pools of enzyme stage 4 used in the two

experiments.

Appendix

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Holiday Period

New Year's Day

Memorial Day Procedure0

5001000150020002500

AP Enzyme Stages

AP Enzyme Stages

mL

Figure 1-1

1 3 5 7 9 11 13 15 17 19 21 23 25 27 290

0.2

0.4

0.6

0.8

1

Alkaline Phoshatase Concentration

Test Tube Fractions

Con

cen

trat

ion

Figure 1-2

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Figure 1-3

0 50 100 150 200 2500

0.5

1

1.5

f(x) = 0.00581013157894737 x + 0.0371578947368419R² = 0.985529550106901

BSA Standard Curve

BSA Concentration (mg/ml)

Abs

orba

nce

(59

5 n

m)

Figure 2-1

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Stg-1 enz Stg-2 enz Stg-3 enz Stg-4 enz0

500

1000

Average Protein Con-centration

AP Stages

Con

cen

trat

ion

mg/

mL

Figure 2-2

Figure 3-1

Absorbance of PNP for Stage #1-4 Enzymes

Stage 1 Stage 2 Stage 3 Stage 40.228 0.228 0.25 0.227

0.298 0.278 0.247

0.295 0.402 0.287 0.256

0.29 0.486 0.316 0.278

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0.286 0.59 0.351 0.3

0.281 0.659 0.383 0.321

0.279 0.739 0.415 0.344

0.277 0.846 0.443 0.368

0.276 0.947 0.474 0.393

0.275 1.056 0.502 0.417

0.274 1.141 0.532 0.438

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

0 20 40 60 80 100 120 140 160 180 2000

0.20.40.60.8

11.2

Absorbance of PNP for Stage #1-4 Enzymes

Stage 1Stage 2Stage 3Stage 4

Time (s)

Ab

sorb

ance

(n

m)

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

Concentration of PNP [ M] at pH 8.0μTime (secs)

Abs 0.0 mM NPP

Abs 0.01 mM PNPP

Ab 0.025 mM NPP

Abs 0.05mM PNPP

Abs0.1 mM PNPP

Abs0.15 mM PNPP

Abs 0.2 mM PNPP

Abs 0.3 mM PPP

Abs 04 mM PNPP

Abs 0.5 mM PNPP

Abs 0.6 mM PNPP

5

47.25897

21

2835538

75

378.0718

336

45.17958

41

1181.474

48

614.3667

297

661.6257

089

992.4385

633

708.8846

881

992.4385

633

1181.474

48

20 0

803.4026

465

1086.956522

1323.251418

1323.251418

1039.697543

1417.769376

1748.582231

1937.618147

1512.287335

1937.618147

40

47.25897

921

1323.251418

1512.287335

1843.100189

2032.136106

1512.287335

2835.538752

2599.243856

3260.869565

2173.913043

2410.207

94

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60 0

1937.618147

2457.466919

2835.538752

2930.056711

2032.136106

3969.754253

3544.423

44

4584.120983

2882.797732

3260.869565

80

47.25897

921

2410.207

94

3166.351607

3544.423

44

3827.977316

2551.984877

5056.710775

4773.156

9

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

47.25897

921

3166.351607

4442.344045

5009.451796

5529.300567

3733.459357

7230.623819

7136.105

86

8837.429112

4300.567108

5529.300567

140 0

3591.682

42

5056.710775

5718.336484

6379.962193

4300.567108

8364.839319

8317.580

34

10349.71645

5151.228733

6285.444234

160

47.25897

921

3969.754253

5623.818526

6427.221172

7183.364839

4584.120983

9404.536862

9404.536862

11814.74

48

5907.372401

7041.587902

180

47.25897

921

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 8.0μ

5.000

20.000

40.000

60.000

80.000

100.000

120.000

140.000

160.000

180.0000.000

2000.000

4000.000

6000.000

8000.000

10000.000

12000.000

14000.000

Concentration of PNP vs Time (s) at pH 8.0

Abs 0.0 mM PNPPAbs 0.01 mM PNPPAbs 0.025 mM PNPPAbs 0.05 mM PNPPAbs 0.1 mM PNPPAbs 0.15 mM PNPPAbs 0.2 mM PNPPAbs 0.3 mM PNPPAbs 0.4 mM PNPPAbs 0.5 mM PNPPAbs 0.6 mM PNPP

Time [sec]

Con

cen

trat

ion

of P

NP

(M

)μ

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

Vo vs. Substrate at pH 8.0S [mM] S [µM] Vo

0 0 00.01 10 22.41425871

0.025 25 32.406157170.05 50 35.10667027

0.1 100 38.617337290.15 150 27.8152849

0.2 200 56.170672430.3 300 54.280313260.4 400 71.833648390.5 500 32.406157170.6 600 38.07723467

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

0 100 200 300 400 500 600 7000

1020304050607080

Vo vs. Substrate at pH 8.0

S[µM]

Vo

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

1/s (mM) 1/s (µM) 1/Vo100 0.1 0.044614458

40 0.04 0.030858333

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20 0.02 0.02848461510 0.01 0.025895105

6.666666667 0.006666667 0.0359514565 0.005 0.017802885

3.333333333 0.003333333 0.0184228862.5 0.0025 0.013921053

2 0.002 0.0308583331.666666667 0.001666667 0.026262411

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

-0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15

-0.02

-0.01

0

0.01

0.02

0.03

0.04

0.05

Lineweaver-Burk Plot at pH 8.0

1/s[µM]

1/V

o

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

Concentration of PNP [ M] at pH 7.0μ

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 mMPNPP

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

13350.78

5

15183.24

6

17801.04

72356.021

1570.681

1570.681

2879.581

2094.241

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40.000

261.78

03141.361

2879.581

21204.18

8

29057.59

2

29319.37

23664.921

2879.581

3141.361

4188.482

4188.482

60.000

0.000

4188.482

3926.702

28272.25

1

40575.91

6

40314.13

64188.482

4188.482

4712.042

6020.942

5497.382

80.000

0.000

5497.382

5235.602

35078.53

4

51570.68

1

50785.34

05235.602

5759.162

6020.942

7853.403

6806.283

100.000

0.000

7591.623

7329.843

41361.25

7

61780.10

5

62041.88

56544.503

7329.843

7329.843

8900.524

8376.963

120.000

0.000

8900.524

8638.743

47120.41

9

71204.18

8

69371.72

87591.623

8900.524

8900.524

10471.20

49947.644

140.000

0.000

10209.42

49947.644

52879.58

1

79842.93

2

78795.81

28900.524

10732.98

4

10471.20

4

12041.88

5

11518.32

5160.000

0.000

11518.32

5

11256.54

5

57853.40

3

88481.67

5

87696.33

5

10209.42

4

12303.66

5

11518.32

5

13612.56

5

13089.00

5180.000

0.000

11780.10

5

12565.44

5

62827.22

5

96858.63

9

96073.29

8

11256.54

5

13612.56

5

12827.22

5

15183.24

6

14921.46

6

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

Time

(secs)

5 20 40 60 80 100 120 140 160 1800

20000400006000080000

100000120000

Concentration of PNP vs Time (s) at pH 7.0 Abs 0.0 mM PNPP

Abs 0.01 mM PNPPAbs 0.025 mM PNPPAbs 0.05 mM PNPPAbs 0.1 mM PNPPAbs 0.15 mM PNPPAbs 0.2 mM PNPPAbs 0.3 mM PNPPAbs 0.4 mM PNPPAbs 0.5 mM PNPPAbs 0.6 mM PNPP

Time [sec]

Con

cen

trat

ion

of P

NP

(M

)μ

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

Vo vs. Substrate at pH 8.0S [mM] S [µM] Vo

0 0 00.01 10 62.82722513

0.025 25 68.810770380.05 50 312.6402393

0.1 100 511.59311890.15 150 502.617801

0.2 200 58.339566190.3 300 73.298429320.4 400 68.810770380.5 500 76.290201940.6 600 77.78608826

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

0 100 200 300 400 500 600 7000

100

200

300

400

500

600

Vo vs. Substrate at pH 7.0

S[µM]

Vo

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

1/s (mM) 1/s (µM) 1/Vo100 0.1 0.015916667

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40 0.04 0.01453260920 0.02 0.00319856510 0.01 0.001954678

6.666666667 0.006666667 0.0019895835 0.005 0.017141026

3.333333333 0.003333333 0.0136428572.5 0.0025 0.014532609

2 0.002 0.0131078431.666666667 0.001666667 0.012855769

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

-0.2 -0.15 -0.1 -0.05 0 0.05 0.1 0.15

-0.02

-0.015

-0.01

-0.005

0

0.005

0.01

0.015

0.02

Lineweaver-Burk Plot at pH 7.0

1/s[µM]

1/V

o

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

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