BarreraBasnetDelgadoLamichhaneShifatuShrestha_Report2_4140_S13

Barrera, Basnet, Delgado, Lamichhane, Shifatu, Shrestha

Juan Barrera, SurajBasnet, Selene Delgado, Mandira Lamichhane, AzeezShifatu, Resha Shrestha

BIOCHEMISTRY LAB 4140

Dr. MesiasPedroza

May 4, 2014

Enzyme Kinetics of Alkaline Phosphatase

Abstract

When isolating enzymes such as Alkaline Phosphatase it is very important to know the rate of

steady flow. The rate of steady flow provides information on how efficient the enzyme is when

binding to a particular substrate. The purpose of the experiment is to test the enzymatic activities

of alkaline phosphatase at different pH levels, and determine the pH at which maximum

efficiency is achieved, by testing the affinity of the enzyme to its substrate. Using a variety of

techniques used in Biochemistry such as salting - in/ salting - out, DEAE – cellulose column

chromatography etc. we were able to successfully isolate the (AP). The AP was then placed in a

gel electrophoresis to separate and analyze the AP based on size and charge. This was followed

by the kinetics portion of the experiment that involved the use of a UV-spectrophotometer. In a

small vial known as a cuvette several different amounts of substrate PNP were added to the

enzyme. The spectrophotometer was used in order to measure the amount absorbed by the

sample as the reaction progressed from p-nitrophenyl (PNP), to Para-nitrophenylphosphate

(PNPP). The data obtained was put together in Michaelis – Menten and Lineweaver – Burk plots

to analyze the enzymatic activity of AP under various different pHs. The Lineweaver – Burk plot

allowed for the successful determination of the affinity of AP to substrate along with the Vmax

at several different concentrations and pH’s.

1


Introduction

The enzyme AP exists in nature in several types of organisms and can easily be extracted from

its location. Its main function is to break down phosphate monoesters via hydrolysis reaction

(Ninfa et al. 2010). AP is an important enzyme utilized by E. coli to convert phosphoryl groups

to inorganic phosphate (Ninfa et al. 2010). The converted phosphoryl groups are used as a

substitute for phosphorous in many important biochemical cell functions (Ming et al. 2010).

During this experiment, AP was located between the outer and inner membranes of the

bacterium, called periplasmic space. The goal of this experiment was to isolate AP from E. coli

via dialysis, salting-in/ salting-out, and DEAE cellulose column chromatography to assess the

purity and the kinetic behavior via SDS-PAGE and graphical analysis of the Lineweaver- Burk

plots. Quantitative and qualitative assessments were conducted to determine the presence and

amount of AP extracted from E. coli. Certain features of AP were used during the isolation

process to differentiate it from other proteins initially present after its release from the

periplasmic space. These features were heat stability, solubility, isoelectric point (pI) of 4.5, and

pH of 8.0. AP also contains two zinc atoms per dimer which able the maximize activity at a

relatively high pH of the hydrolysis of phosphate esters (Ninfa et al. 2010). Below is the equation

that represents the hydrolysis of phosphate monoesters:

Figure 1: AP phosphate chemistry

The figure shows Alkaline Phosphate reaction: Phosphate monoester binds with water to

produce alcohol and alkaline phosphate.

(http://www.worthington-biochem.com/bap/default.htm) l

Purification and Denaturation of Alkaline Phosphatase

2

http://www.worthington-biochem.com/bap/default.htm


In order to study the function and structure of AP, it first must be isolated from E. coli and

purified. The isolation step required the liberation of AP from the periplasmic space by breaking

down the outer membrane with lysozyme enzyme leaving the inner membrane and its content

intact (Ninfa et al. 2010). While AP was released, DNA present in the extraction was broken

down using an enzyme called DNAse. Dialysis of the enzyme (2) allows the diffusion of

molecules through a semi-permeable membrane of a dialysis bag based on their size and

concentration (Ninfa et al. 2010). The denaturation of unwanted proteins present in the AP

extraction from E. coli is possible by heating up the extraction at 80°C. The heat stability of AP

will maintain the AP protein intact and preserved in solution after centrifugation.

Figure 2: Dialysis Diagram

Dialysis of the enzyme allows the movement of molecules through the dialysis bad which acts as

a semi-permeable membrane.

(http://www.spectrumlabs.com/dialysis/Fund.html)

Ammonium sulfate precipitation commonly called salting-in/salting- out was used to purify the

extracted AP. The addition of ammonium sulfate to the extraction allows the separation of

proteins based on their solubility at high salt concentration (Voet et al. 2012). With the addition

of salts, AP will fall out of solution and will be present in pellets formed after centrifugation.

In order to further electrostatically separate, a positively charged anion exchanger by

diethylaminoethyl (DEAE) groups linked to a cellulose column allows the purification of AP

3

http://www.spectrumlabs.com/dialysis/Fund.html


based on its pI and pH. The eluting step allows the bound AP to be removed via addition of a

higher concentration of negatively charged ions and thus out-competing the AP (Ninfa et al

2010).

Quantification of Alkaline Phosphatase

The quantitative analysis of AP concentration was effectuated with a colorimetric test to

positively identify its presence in samples. The collected fractions from the DEAE-cellulose

chromatography step can be qualitatively assessed using Bradford dye-binding method. When

mixing Sigma-Fast BCIP-NBT with a small volume of the eluted fraction sample, a color change

can be elicited that refers to the enzymatic activity of AP present in the sample tested. The

amount of light absorbed by the sample using a UV spectrophotometer at 595 nm provides data

that can be used to construct a standard bovine serum albumin (BSA) curve. The solution used to

perform the colorimetric test was made by diluting BSA with Coomassie Brilliant Blue dye

(Ninfa et al. 2010).

BCIP-NBT

The molecule 5-Bromo-4-chloro-3-indolyl phosphate (BCIP, X-phosphate, XP) is an synthetic

chromogenic substrate used for the sensitive colorimetric detection of alkaline phosphatase

activity. It is, used in immune-blotting, in situ hybridization, and immunohistochemistry, in

combination with nitro blue tetrazolium chloride (NBT). Alkaline phosphatase hydrolyses BCIP

to 5-bromo-4-chloro-3-indole and inorganic phosphate. 5-bromo-4-chloro-3-indole is oxidized

by atmospheric oxygen to form the blue dye 5,5′-dibromo-4,4′-dichloro-indigo. It is also

oxidized by nitro-blue tetrazolium (NBT), which forms an insoluble dark blue diformazan

precipitate after reduction. Alkaline phosphatase is commonly conjugated to secondary

antibodies. (http://www.sigmaaldrich.com/catalog/product/sigma/b1026?lang=en&region=US)

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique widely

used in biochemistry, molecular biology, and genetics to separate proteins based on their charge

and mass (Ninfa et al. 2010). The charge of the protein samples was normalized by treating the

proteins with sodium dodecyl sulfate (SDS), which coat the protein with negative charges (Ninfa

4


et al. 2010). Also, the presence of glycerol in SDS is used to help the sample loaded in the gel

sink to the bottom of the well based on its density. SDS-PAGE was used to verify the purity of

AP protein extracted and calculate its molecular weight.

Enzyme Kinetics

After the isolation, purification and assay of enzyme, enzyme kinetics can be studied which is

characterized by the steady state kinetic parameters as Km, Vmax, Kcat, and Kcat/ Km. These

constants provide information on enzyme’s response to substrates. PNPP is the single substrate

used in this experiment, the kinetics of one-substrate reactions is E + S ↔ ES ↔ E + P, where E

is the enzyme, S is the substrate, ES is the enzyme-substrate complex, and P is the product.

There are several assumptions that can be made based on the one substrate equation. The

Michaelis-Menton equation is based on the assumption that ES is in equilibrium with E and S.

Another assumption was that the ES complex is in steady state which translates to the rate of

formation of ES equals the rate of breakdown of ES, where k1 and k2 are >>> k2. From the

assumptions, it can be determined that [ES] remains in essence constant during the reaction. For

the given concentration of AP, the Michaelis-Menton equation is:

V = (Vmax [S]) / (KM + [S]), where V is the velocity of the catalyzed reaction at any time,

Vmax is the maximum velocity of the total enzyme population when the enzyme is saturated

with substrate.

Figure 3. Michaelis-Menton Plot.

Plot of kinetic data indicating rate of

(http://depts.washington.edu/wmatkins/kinetics/michaelis-menten.html)

5

http://depts.washington.edu/wmatkins/kinetics/michaelis-menten.html


Km is the Michaelis constant whose value is the substrate concentration when rate of reaction is

half maximal. It is the measure of binding affinity of an enzyme to a substrate. Higher the value

of Km, the lower the enzyme has the affinity to a substrate. Catalytic constant (kcat) is s the

maximum turnover number and is equal to k2. The catalytic efficiency of an enzyme can be

expressed by the equation kcat / Km.

The Michaelis-Menton graph is a hyperbola and is therefore challenging to find the exact value

of Vmax and Km . Therefore, it is modified to produce a linear line to easily determine Vmax

and Km. This new graph is referred to as a Lineweaver-Burk equation.

Figure 4. Lineweaver-Burk Plot.

Double-reciprocal plot of kinetic data, showing the meaning of the axis intercepts and slope.

(http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html)

The graph consist of y= 1/Vo and x= 1/[S]. From this graph, the kinetic parameters can be easily

and accurately determined. Km/Vmax is the slope, the y-intercept is 1/Vmax and the x-intercept

is -1/Km.

Para-nitrophenylphosphate (PNPP) was used as the substrate yielding to PNP when in contact

with the AP enzyme. The kinetic behavior of AP was studied under three different pH values 7.0,

8.0, and 9.0. It was achieved by plotting the Lineweaver – Burk using the data collected from the

6

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html


enzyme kinetic experiment. The values of Km and Vmax were estimated from the equation

given from the Lineweaver-Burk plots at each pH.

The purpose of the experiment is to test the enzymatic activities of alkaline phosphatase at

different pH levels, and determine the pH at which maximum efficiency is achieved. This is done

by testing the affinity of the enzyme to its substrate, and the turnover number of products per

substrate .Success is achieved when the enzyme is proven to function the best on its optimal pH

of 8.

Methods

The following procedure for isolation, purification, and quantification was done as described in

the lab manual with modifications (Ninfa et al. 2010).

Day 1: Isolation of Alkaline Phosphatase

All the samples were kept on ice to prevent their degradation. 20 ml of hydrated E. coli K-12

cells were transferred to a 50 ml polycarbonate centrifuge tube. The suspension was warmed

with hands for few minutes. Once warmed, 0.2 ml of lysozyme was added to the centrifuge tube

containing the E. coli cells and gently swirled. The tube was then incubated at room temperature

for 20 minutes. After incubation, 0.2 ml of DNAse I at 10,000 units/ml-1 was added to the

solution and gently swirled. The solution was incubated at room temperature for 5 minutes.

Then, 0.2 ml of 0.1 M EDTA was added to the solution, gently swirled and incubated for 10

minutes. During the incubation period, an appropriate length of dialysis tubing was cut and

soaked into a beaker of deionized water. Once the incubation was completed, 0.2 ml of 1 M

MgSO4 was added to the solution, swirled and incubated for 20 minutes. Then, the centrifuge

tube containing the solution was centrifuged at 10,000 rpm at 22°C. Slowly, the tube was

removed from the rotor. The supernatant was collected and transferred to a clean and empty tube.

The pellets were discarded. The volume of the supernatant was measured and recorded as part of

the results. 0.5 ml of the supernatant was collected and stored at -20°C for enzyme assay and

quantification. Two knots were tied at one end of the dialysis tubing to form a dialysis bag. The

remaining supernatant was carefully transferred to the dialysis bag. A second pair of knots was

tied to the other end of the dialysis bag leaving sufficient space to let Stage I enzyme expand

during dialysis. In a beaker, 500 ml of 10 mMTris-HCl (pH7.4) and 10 mM MgSO4 dialysis

7


buffer was prepared. The dialysis bag was placed in the beaker containing the dialysis buffer.

The beaker was labeled with the description, the date, and the name of each group member and

was placed in the refrigerated. The buffer was changed two and four days after it was made.


Stage I Enzyme solution was collected from the dialysis bag, measured and placed into a

centrifuge tube. The solution placed in a tube was heated in an 80ºC water bath for 15 minutes.

After heating the solution, the solution was placed under cold water for few seconds. Stage I

Enzyme was centrifuged for 20 minutes at 10,000 rpm at 4ºC. An appropriate length of dialysis

tubing was cut and soaked in deionized water. Once the centrifugation was completed, the tube

containing the solution was carefully removed from the rotor without disturbing the pellets. The

supernatant was collected and measured. The volume of the supernatant considered as Stage II

Enzyme was recorded as part of the results. 0.5 ml of Stage II Enzyme was stored for enzyme

assay and quantification. Ammonium sulfate was slowly added to the remaining solution and

stirred until complete dissolution of the ammonium, sulfate powder. The amount of ammonium

sulfate powder was determined according to the volume of Stage II Enzyme with the following

equation:

Volume of supernatant (ml) X 0.603 g/1ml = quantity of (NH4)2SO4 (g)

The tube containing the solution was centrifuged for 30 minutes at 18,000 rpm at 4ºC. Once

completed, the supernatant was decanted and discarded. The conserved pellets were carefully

suspended into 0.5 ml of 10mM Tris-HCl (pH7.4) and 10mM MgSO4 dialysis buffer. A dialysis

bag was prepared from the dialysis tubing soaked in deionized water. The suspended pellets were

transferred to the dialysis bag. After leaving enough space for the solution to expand, a knot was

tied on the open end of the dialysis bag. This latter was placed into a beaker with 10mM Tris-

HCl (pH7.4) and 10mM MgSO4 dialysis buffer and kept in the refrigerator. The dialysis buffer

was changed twice before the next laboratory session.

8



The dialyzed solution was retrieved, measured and poured into a 50 ml polycarbonate centrifuge

tube. The volume of the solution referred as Stage III Enzyme was recorded as part of the results.

0.5 ml of Stage III Enzyme was removed and stored for enzyme assay and protein quantification.

DEAE-Cellulose Column Chromatography

A DEAE chromatography column was prepared using 2.5 cm of a mixed DEAE resin and buffer

A solution. A faculty member of University of Houston-Downtown prepared this DEAE-

cellulose solution. 20 clean test tubes (13x100 mm) were each marked at 1.5 ml level. Stage III

Enzyme was poured into the column. In 6 different test tubes, 1.5 ml of the Stage III Enzyme

was collected from the column. Next, 5 ml of buffer A was poured in the column and collected in

6 test tubes to 1.5 ml level marked. 1.0 ml of eluting buffer B was poured into the column and

collected into the test tubes. This last step was repeated 8 times.

Spot test

The activity of AP was tested in the 20 different fractions collected from the DEAE

chromatography step. On a small piece of Parafilm, 10 μL of solution from each fraction was

placed in columns according to their buffer solutions. Next, 5 μL of Sigma-Fast BCIP was added

to each 10 μL samples on the Parafilm. 5 minutes later, the color change of each sample from the

Parafilm was recorded. The fractions showing a positive result for the spot test were collected

into a single tube and labeled Stage IV Enzyme.

Day 4: Quantification of Alkaline Phosphatase

The spectrophotometer was set at 595 nanometer (nm) 15 minutes before taking any

measurement. The total volume of Working Bradford reagent required for the samples was

calculated using the following equation:

(# of wells) [0.2 ml (Bradford Reag.)] + 2.0 ml =Total Vol. of Bradford Reag.

The total volume of Bradford reagent was increased to an even number. In each tube, 0.2 ml of

Bradford reagent was added and carefully mixed. Stage I, II, III, IV enzyme samples were placed

9


in different wells of the 96 wells immunology plate used to quantify AP. The total volume of

each well was 50 μL. Once all the samples were put in the wells, the plate was incubated for 10

minutes at room temperature before taking any measurement. The absorbance was measured at

595 nm and recorded. The absorbance readings were used to construct a plot for a standard BSA

curve. The equation derived from the standard BSA curve was used to estimate the final

concentration for each Stage Enzyme.

Gel Electrophoresis

The gel apparatus was assembled with two gels and placed in the chamber filled with enough

10X SDS-PAGE running buffer to run both gels. The same buffer was added to the mini tank

between the two gels.

Then, Stage I, II, III, IV Enzyme solution, a commercial AP sample, and a protein ladder were

prepared in separate Eppendorf tubes. In each tube, 20 μL of each Stage Enzyme with 20 μL of 2

X SDS-PAGE buffers were added. In one of the sample tube, 20 μL of commercial AP with 20

μL of 2X sample buffer were added. All the tubes were heated for 5 minutes in a hot water bath

and then cooled by running cold water on the samples. According to the following scheme, 7 μL

of protein ladder, 20 μL of each sample tube including the pure AP sample were loaded into the

wells of the gel:

After placing the lid on the chamber, the electrical leads were plugged to the power supply with

the proper polarity and set to 180 volts. The gel electrophoresis was run for about 1 hour until the

dye present in the samples reached the bottom of the gel. The power cords were disconnected

and the tank lid was removed. The inner chamber was disassembled to remove the gel from the

two glass plates. The gel was stained for 2 days by placing it in a small container filled with

Coomassie Blue stain. The box was covered with Parafilm. Two days later, the staining gel

solution was poured back to its original bottle while the gel was rinse once with deionized water.

The gel was then placed into a distaining solution for two days.

Enzyme Kinetics of Alkaline Phosphatase

The reference was prepared with 3 ml of 0.2 M Tris-HCl (pH 8.0) into a cuvette to measure the

absorbance of 50 µM PNPP at 410 nm. The extinction coefficient was calculated using the

10


collected absorbance measurement from the reference sample (A). The following Beer- Lambert

law equation was used to estimate the extinction coefficient:

A = Ԑ Cl

Another reference cuvette was prepared using 1.5 ml of 1 mM PNPP and 1.5 ml of 0.2M Tris-

HCl (pH 8.0) into a cuvette. The absorbance was measured at 410 nm. According to Table 2, 7

samples were prepared to analyze the kinetics of AP. This step was repeated three times using

0.2 M Tris- HCl buffer at pH 6.8 and pH 8.8.

Results

The following results were obtained from the gel electrophoresis test that performed on all 4

stages of enzyme.

Figure 5. Gel Electrophoresis.

Stained gel after poly acrylamide gel electrophoresis. The lanes included the different stage

enzymes. Starting from the right is the molecular weight marker followed by the different stage

enzymes, i.e. MW Ladder, Stage 1, Stage 2, Stage 3, Stage 4 and Commercial AP.

11


Table 1. Absorbance values of Stage 4 enzyme

Absorbance Absorbance ul Avg Abs Conc St40.332 0.347 12.5 0.3395 65.28846 Avg Gr1 68.076920.37 0.367 25 0.3685 70.865380.611 0.503 12.5 0.557 107.1154 Avg Gr2 121.77880.704 0.715 25 0.7095 136.44230.982 0.763 12.5 0.8725 167.7885 Avg Gr3 151.73080.731 0.68 25 0.7055 135.67310.876 0.809 12.5 0.8425 162.0192 Avg Gr4 158.36540.864 0.745 25 0.8045 154.7115

The absorbance values for each group were averaged out and used to calculate the total enzyme

concentration.

Total Enzyme

=0.025798

12


Table 2. Absorbance Zero-corrected at different concentrations and time at pH 7.

Absorbance zero-out

0.0mM

PNPP

0.01mM PNPP

0.025mM PNPP

0.05mM

PNPP

0.1mM

PNPP

0.15mM

PNPP

0.2mM PNPP

0.25mM

PNPP

0.3mM PNPP

0 0.000 0.000 -0.001 0.001 0.001 0.002 0.002 0.003 0.0075 0.000 0.001 0.000 0.002 0.001 0.003 0.010 0.005 0.00920 0.000 0.002 0.001 0.003 0.002 0.003 0.011 0.005 0.00940 0.001 0.002 0.002 0.003 0.003 0.004 0.012 0.006 0.01060 0.000 0.004 0.002 0.004 0.004 0.005 0.012 0.007 0.01080 0.000 0.005 0.004 0.004 0.004 0.006 0.013 0.008 0.011100 0.000 0.006 0.005 0.005 0.005 0.007 0.014 0.009 0.012120 0.000 0.007 0.006 0.006 0.006 0.007 0.015 0.010 0.012140 0.000 0.008 0.007 0.006 0.007 0.008 0.016 0.011 0.013160 0.000 0.009 0.008 0.007 0.008 0.009 0.017 0.012 0.014180 0.000 0.010 0.009 0.008 0.009 0.010 0.018 0.013 0.015

Graph 1: The absorbance values for each PNP product formed as a function of time pH7.

The absorbance values for each PNP product formed as a function of time. Each concentration of substrate is represented by a different color on the right side of the graph.

13

0 20 40 60 80 100 120 140 160 180 200

-0.005

0.000

0.005

0.010

0.015

0.020

Absorbance vs Time pH7

0.0mM PNPP0.01mM PNPP0.025mM PNPP0.05mM PNPP0.1mM PNPP0.15mM PNPP0.2mM PNPP0.25mM PNPP0.3mM PNPP

Time (sec)

Abso

rban

ce Z

ero

Corr

ecte

d


1) Abs zero corrected

0.0 mM PNPP

0.01 mM PNPP

0.025 mM PNPP

0.05mM PNPP

0.1mM PNPP

0.15mM PNPP

0.2mM PNPP

0.25mM PNPP

0.3mM PNPP

0 0 0 0 0.01 0.002 0.003 0.004 0.005 0.0095 0 0.007 0.006 0.016 0.01 0.008 0.009 0.015 0.015

20 0 0.014 0.016 0.022 0.016 0.014 0.019 0.014 0.02140 0 0.023 0.026 0.031 0.024 0.019 0.025 0.024 0.03160 0 0.035 0.035 0.04 0.038 0.03 0.036 0.032 0.04180 0 0.044 0.046 0.051 0.048 0.042 0.045 0.04 0.048

100 0 0.054 0.056 0.06 0.058 0.053 0.054 0.047 0.055120 0 0.062 0.066 0.071 0.069 0.061 0.063 0.054 0.063140 0 0.07 0.075 0.081 0.08 0.07 0.072 0.062 0.071160 0 0.078 0.083 0.091 0.09 0.08 0.082 0.07 0.078180 0 0.084 0.092 0.101 0.1 0.091 0.092 0.078 0.086Table 3. Absorbance Zero-corrected at different concentrations and time at pH 8.

Graph 2: The absorbance values for each PNP product formed as a function of time pH 8

The absorbance values for each PNP product formed as a function of time. Each concentration of substrate is represented by a different color on the right side of the graph.

14

0 20 40 60 80 100 120 140 160 180 2000

0.02

0.04

0.06

0.08

0.1

0.12

Absorbance vs Time pH 8

0.0 mM PNPP0.01 mM PNPP0.025 mM PNPP0.05mM0.1mM0.15mM0.2mM0.25mM0.3mM

Time (sec)

Abso

rban

ce Z

ero-

Corr

ecte

d


Table 4. Absorbance Zero-corrected at different concentrations and time at pH 9.

Time(Seconds)

0.0 mM PNPP

0.01 mM PNPP

0.025 mM PNPP

0.05mM PNPP

0.1mM PNPP

0.15mM PNPP

0.2mM PNPP

0.25mM PNPP

0.3mM PNPP

0 0.000 0.001 0.003 0.001 0.002 0.003 0.006 0.006 0.0075 0.000 0.008 0.014 0.011 0.011 0.010 0.012 0.013 0.018

20 0.000 0.026 0.029 0.024 0.022 0.015 0.014 0.044 0.02640 0.000 0.040 0.055 0.049 0.034 0.032 0.014 0.073 0.04360 0.000 0.057 0.080 0.073 0.060 0.054 0.015 0.106 0.06880 0.000 0.071 0.103 0.100 0.085 0.076 0.017 0.143 0.089

100 0.000 0.083 0.124 0.125 0.107 0.101 0.019 0.182 0.115120 0.000 0.093 0.144 0.148 0.128 0.127 0.024 0.219 0.139140 0.000 0.102 0.163 0.170 0.149 0.154 0.031 0.257 0.166160 0.000 0.110 0.179 0.190 0.173 0.179 0.037 0.299 0.192180 0.000 0.116 0.195 0.209 0.195 0.204 0.042 0.343 0.218

Graph 3: The absorbance values for each PNP product formed as a function of time pH9.

The absorbance values for each PNP product formed as a function of time. Each concentration

of substrate is represented by a different color on the right side of the graph.

15

0 20 40 60 80 100 120 140 160 180 2000.0000.0500.1000.1500.2000.2500.3000.3500.400

Absorbance vs Time pH 9

0.0 mM PNPP0.01 mM PNPP0.025 mM PNPP0.05mM PNPP0.1mM PNPP0.15mM PNPP0.2mM PNPP0.25mM PNPP0.3mM PNPP

Time (sec)

Abso

rban

ce Z

ero-

Corr

ecte

d


Time (Seconds)

Stage 1

absorbance zero-out

Stage 2

absorbance zero-out2

Stage 3

absorbance zero-out3 Stage 4

absorbance zero-out4

0 0.042 0 0.042 0 0.042 0 0.042 05 0.06 0.018 0.048 0.006 0.044 0.002 0.047 0.005

20 0.078 0.036 0.052 0.01 0.044 0.002 0.053 0.01140 0.104 0.062 0.055 0.013 0.048 0.006 0.067 0.02560 0.129 0.087 0.057 0.015 0.051 0.009 0.077 0.03580 0.152 0.11 0.064 0.022 0.054 0.012 0.091 0.049

100 0.176 0.134 0.07 0.028 0.057 0.015 0.104 0.062120 0.198 0.156 0.075 0.033 0.06 0.018 0.114 0.072140 0.219 0.177 0.08 0.038 0.063 0.021 0.124 0.082160 0.239 0.197 0.086 0.044 0.066 0.024 0.134 0.092180 0.259 0.217 0.093 0.051 0.069 0.027 0.145 0.103

Table 5. Absorbance Zero-corrected of Stages 1-4 at pH 8.

Graph 4: The absorbance values for stages 1-4 at the optimal pH of 8

The absorbance values for each Stage of AP product as a function of time. Each stage of enzyme is represented by a different color on the right side of the graph.

Table 6. Product formed at various time at pH 7.

16

0 20 40 60 80 100 120 140 160 180 2000

0.05

0.1

0.15

0.2

0.25

Stages 1-4 Absorbance vs time pH 8

Stage 1Stage 2Stage 3Stage 4

Time (sec)

Abso

rban

ce Z

ero-

Corr

ecte

d


Graph 5. The product formed as a function of time at pH 7.

The amount of product formed for each concentration can be seen on the y-axis. The different

concentrations of substrate are indicated by different shapes and colors.

17

0 20 40 60 80 100 120 140 160 180 200-0.5

0

0.5

1

1.5

2

2.5

3

Product formed vs Time pH 7

0.0mM PNPP0.01mM PNPP0.025mM PNPP0.05mM PNPP0.1mM PNPP0.15mM PNPP0.2mM PNPP0.25mM PNPP0.3mM PNPP

Time

Prod

uct F

orm

ed



3) Product formed (uM)

0.0 mM PNPP

0.01 mM PNPP

0.025 mM PNPP 0.05mM 0.1mM 0.15mM 0.2mM 0.25mM 0.3mM

0 0 0 01.28534704

40.2570694

10.385604

10.51413

90.642673

51.15681

2

5 00.89974293

10.77120822

6 2.056555271.2853470

41.028277

61.15681

21.928020

61.92802

1

20 01.79948586

1 2.056555272.82776349

62.0565552

71.799485

92.44215

91.799485

92.69922

9

40 02.95629820

13.34190231

43.98457583

5 3.08483292.442159

43.21336

83.084832

93.98457

6

60 04.49871465

34.49871465

35.14138817

54.8843187

73.856041

14.62724

94.113110

55.26992

3

80 05.65552699

25.91259640

16.55526992

36.1696658

15.398457

65.78406

25.141388

26.16966

6

100 06.94087403

67.19794344

57.71208226

27.4550128

56.812339

36.94087

46.041131

17.06940

9

120 07.96915167

18.48329048

8 9.12596401 8.8688946 7.8406178.09768

6 6.9408748.09768

6

140 08.99742930

69.64010282

810.4113110

510.282776

38.997429

39.25449

97.969151

79.12596

4

160 010.0257069

410.6683804

6 11.696658111.568123

410.28277

610.5398

58.997429

310.0257

1

180 010.7969151

7 11.825192812.9820051

412.853470

411.69665

811.8251

910.02570

711.0539

8


18


0 20 40 60 80 100 120 140 160 180 2000

2

4

6

8

10

12

14

Product Formed vs time pH 8

0.0 mM PNPP0.01 mM PNPP0.025 mM PNPP0.05mM0.1mM0.15mM0.2mM0.25mM0.3mM

Time (sec)

Prod

uct F

orm

ed

The amount of product formed for each concentration can be seen on the y-axis. The different

concentrations of substrates are indicated by different shapes and colors.


Time (Sec)

0.0 mM PNPP

0.01 mM PNPP

0.025 mM PNPP

0.05mM PNPP

0.1mM PNPP

0.15mM PNPP

0.2mM PNPP

0.25mM PNPP

0.3mM PNPP

0 0 0.055803571 0.167410714 0.055803571 0.111607143 0.167410714 0.334821429 0.334821429 0.3906255 0 0.446428571 0.78125 0.613839286 0.613839286 0.558035714 0.669642857 0.725446429 1.004464286

20 0 1.450892857 1.618303571 1.339285714 1.227678571 0.837053571 0.78125 2.455357143 1.45089285740 0 2.232142857 3.069196429 2.734375 1.897321429 1.785714286 0.78125 4.073660714 2.39955357160 0 3.180803571 4.464285714 4.073660714 3.348214286 3.013392857 0.837053571 5.915178571 3.79464285780 0 3.962053571 5.747767857 5.580357143 4.743303571 4.241071429 0.948660714 7.979910714 4.966517857

100 0 4.631696429 6.919642857 6.975446429 5.970982143 5.636160714 1.060267857 10.15625 6.417410714120 0 5.189732143 8.035714286 8.258928571 7.142857143 7.087053571 1.339285714 12.22098214 7.756696429140 0 5.691964286 9.095982143 9.486607143 8.314732143 8.59375 1.729910714 14.34151786 9.263392857160 0 6.138392857 9.988839286 10.60267857 9.654017857 9.988839286 2.064732143 16.68526786 10.71428571180 0 6.473214286 10.88169643 11.66294643 10.88169643 11.38392857 2.34375 19.140625 12.16517857


19


The

amount of product formed for each concentration can be seen on the y-axis. The different

concentrations of substrates are indicated by different shapes and colors.

Table 9. Product formed by various Stages 1-4 at pH 8.

Time (Seconds) Stage 1 Stage2 Stage 3 Stage 40 0 0 0 05 2.31362468 0.771208226 0.25706941 0.642673522

20 4.62724936 1.285347044 0.25706941 1.41388174840 7.96915167 1.670951157 0.77120823 3.21336760960 11.1825193 1.928020566 1.15681234 4.49871465380 14.1388175 2.827763496 1.54241645 6.298200514

100 17.2236504 3.598971722 1.92802057 7.969151671120 20.0514139 4.241645244 2.31362468 9.254498715140 22.7506427 4.884318766 2.69922879 10.53984576160 25.3213368 5.655526992 3.0848329 11.8251928180 27.8920308 6.555269923 3.47043702 13.23907455

Graph 8. The product formed as a function of time of Stages 1-4 enzymes at optimal pH 8.

20

0 20 40 60 80 100 120 140 160 180 2000

5

10

15

20

25

Product formed vs time pH 9

0.0 mM PNPP0.01 mM PNPP0.025 mM PNPP0.05mM PNPP0.1mM PNPP0.15mM PNPP0.2mM PNPP0.25mM PNPP0.3mM PNPP

Time (sec)

Prod

uct F

orm

ed


0 20 40 60 80 100 120 140 160 180 2000

5

10

15

20

25

30 Product formed vs time pH 8

Stage 1Stage 2Stage 3Stage 4

Time (sec)

Prod

uct F

orm

ed

The amount of product formed for each Stage of AP can be seen on the y-axis. The different

stages of AP are indicated by different shapes and colors.

Table 10. Substrate Concentration vs. Initial Velocity

[S] (µM) Vo μM/s

101.37614678

9

251.37614678

9

500.91743119

3100 1.22324159

1501.07033639

1200 1.22324159250 1.22324159

3000.91743119

3

Graph 9: Michaelis-Menten plot pH7.

21


The plot in indicated by the PNPP concentration on the x-axis and reaction velocity on the y-axis.

Table 11. Double reciprocal kinetics data.

1/[S] 1/V00.1 0.726666667

0.04 0.7266666670.02 1.090.01 0.8175

0.0066667 0.9342857140.005 0.81750.004 0.8175

0.0033333 1.09

22

0 50 100 150 200 250 300 3500

0.20.40.60.8

11.21.41.6

Michaelis-Menten pH7

[S] PNPP (uM)

Vo (u

M*s

-1)


Graph 10: Lineweaver-Burk plot pH 7.

Y-axis represents 1/Vo of each absorbance of PNP as a function of 1/concentration (x-axis) of

the substrate, PNPP. The km value was obtained by -1/ x-intercept and the Vmax was obtained

by 1/y-intercept.

Vmax = 6.75676

Km = 16.67

Kcat = 261.9102

Efficiency = 15.711

Table 12. Substrate Concentration vs. Initial Velocity

Sub [uM] Vo

0 0

109.89717

2

2511.0539

8

5010.9254

5

10011.5681

2

23


15010.6683

8

20010.6683

8

2508.09768

6

3009.12596

4

Graph 11: Michaelis-Menten plot pH8.

The plot in indicated by the PNPP concentration on the x-axis and reaction velocity on the y-

axis.

Table 13. Double reciprocal kinetics data.

1/Sub [] 1/Vo

0.1 0.1010

24

0 50 100 150 200 250 3000

10

20

30

40

50

60

70

80

Michaelis-Menten pH8

[S] PNPP (uM)

Vo (u

M *

S-1

)


39

0.040.0904

65

0.020.0915

29

0.010.0864

440.006666

670.0937

35

0.0050.0937

35

0.0040.1234

920.003333

330.1095

77

Graph 12: Lineweaver-Burk plot pH 8.



by 1/y-intercept.

Vmax = 62.5

Km= 0.025

25


Kcat = 2422.6684

Efficiency = 96906.73696

Table 14. Substrate Concentration vs. Initial Velocity pH 9

[S] µM Vo0 0

10 6.02678571425 10.1004464350 11.04910714

100 10.26786150 10.82589200 1.674107250 18.41518300 11.16071

Graph 13: Michaelis-Menten plot pH 9.

The plot in indicated by the PNPP concentration on the x-axis and reaction velocity on the y-axis.

26

0 50 100 150 200 250 300 35002468

101214161820

Michaelis-Menten pH 9

[S] PNPP (uM)

Vo (u

M *

s-1)


Table 13. Double reciprocal kinetics data pH9

1/[S] 1/V00.1 0.165925926

0.04 0.0990055250.02 0.0905050510.01 0.097391277

0.006666667 0.0923711580.005 0.5973333840.004 0.054303026

0.003333333 0.089600034

Graph 14: Lineweaver-Burk plot pH 9



by 1/y-intercept.

Vmax = 200

27


Km = 10Kcat = 7752.53896Efficiency = 775.253896

Discussion

Many methods of biochemistry were used in this experiment. Each technique was specifically

used to result in the extraction and purification of Alkaline Phosphate. The Alkaline Phosphate

was extracted from K-12 E. coli, which is a mutated strand of E. coli that expresses production of

Alkaline Phosphate readily. Ion exchange chromatography was used to isolate alkaline

phosphatase from the E.coli k-12 cells. In this method, the protein of interest sticks to the column

and the rest of the proteins elute out of the column. The proteins of interest are then removed

with a salt. After that Spot Test was used to determine which fractions have enzyme activity. A

dye name Sigma-Fast BCPI was used to indicate Alkaline Phosphate enzyme activity. Fractions,

which had shown enzyme activity, were fractions 15-19 which were expected. Fractions 1-14

and 20 tested negative for Alkaline Phosphate by showing no color change. Fractions 18 and 19

were light purple when compared to fraction 15, 16, and 17. This indicates that there was more

enzyme activity occurring.

Protein Quantification was done by using a Bradford assay kit and measuring the absorbance of

each stage of protein. A standard was created as contrast to the resulting stages. After the protein

was isolated, a polyacrylamide gel electrophoreses was run. The gel is composed of two different

gels a stacking gel and a running gel. The gel separated enzymes on the basis of their different

sizes. Small enzymes moved faster than larger proteins, resulting in a series of ‘bands’. Each

band contained an enzyme of a specific size. We compared them with the standards used. Stage 1

enzyme looked almost like the ladder. There was a large amount of dye in certain area of gel.

Because of the dye it is difficult to determine how far the enzyme traveled. It is also difficult to

determine how far the other enzymes traveled. The bands are not very clear.

After the gel electrophoreses, the absorbance of the alkaline phosphatase mixed with nitrophenyl

(PNP) and p-nitrophenyl phosphate (PNPP) was obtained using a spectrophotometer. For this

different pH( 6.8, 8.0,8.8) of Tris- HCL was added to the mixture.

28


Enzyme kinetics were calculated based on the results from the UV-spectrophotometer

absorbance values. The values for the zero corrected absorbance’s for pH 7,8, and 9 can be seen

in tables 2,3 and 4 respectively. From these values, an absorbance vs time graph was conducted

for each different pH value. The graphs 1,2 and 3 show similar patterns of absorbance’s over

time. The absorbance’s always increase over time and show an upward trend. However it can be

seen that for different pH values, different amounts of PNP show different results. For example

in pH 7 and pH 8, the greatest absorption value came from 0.2mM PNPP while in pH 9, the

greatest absorption came from 0.25mM. This same trend can be viewed on the product formed

vs time graphs. The concentrations that contained the greatest absorption produced the most

product over time. The Michaelis-Menton plot in graph 9 for pH 7 indicates a relatively constant

line that could be an indication that the velocity of the reaction was not dependent on the

concentration of PNPP. The Lineweaver-Burke plot seen in graph 10 indicates the maximum

velocity at 6.75 uM/s. This value indicates the maximum velocity at which the reaction will

occur at pH 7. It also indicates the important values of kcat, and km that were essential to

determine the efficiency of the enzyme that was noted at 15.711 uM/s. The efficiency was

obtained by dividing the turnover (kcat) by the affinity (km). In pH 8, the Michealis-Menton plot

also indicates a constant line of absorption around the 60 to 70um/s range. The Lineweaver-

Burke plot seen in graph 12 indicates the x-intercept as increased to the left. This is a clear

indication of greater enzyme affinity to substrate as the Km decreases. The affinity calculation

reveals a massive increase when compared to pH7 and is noted as 9.69x104 uM/s.

The Michaelis-Menton graph for pH 9 again shows consistent numbers as time increases. The

Lineweaver-Burke plot as seen in graph 14 indicates the efficiency as 775.25 uM/s. From

comparison of all the values clear conclusions can be obtained. It is pretty apparent that the

enzyme is the most efficient at pH 8 followed by pH 9 and pH 7 showed the worst results. There

was also an absorbance plot at pH8 obtained for Stages 1-4 enzymes. Stage 1 showed the

greatest amount of absorption while stage 3 showed the lowest. Stage 1 could possibly have

showed the highest absorption due to the presence of other proteins in solution.

Based on the fact that AP is an enzyme that works best under alkaline or basic conditions our

results make sense. The pH 8 and pH9 are basic environments under which the enzyme works

best. It was noted before the experiment that the optimal pH for AP was at pH 8 and our results

29


confirm this. In the theory behind the experiment it was noted that as (km) values decreased, the

affinity increases. This was clearly confirmed in this experiment as the lowest km values

ultimately lead to high efficiency numbers. The higher the pH, the higher the Vmax was also

noted in this experiment.

One of the ways to improve the overall experiment is to have clear and concise written

instructions of every step involved in this experiment. It becomes increasingly difficult to follow

the steps when they are simply written on the board the day of the experiment. Another aspect

that would improve the experiment is having knowledge beforehand of the theory behind

kinetics. The theory of kinetics was not covered in lecture until after the experiment was

completed. We were left questioning the exact purpose of the experimental procedure and this I

believe affected our performance. Lastly I believe it is essential for groups to consist of fewer

team members to adequately allow every member to be involved.

30


Works Cited

BioRad. SDS- PAGE Molecular Weight Standards Low Range - Catalog # 161-0304

Ming, L., Yong-Chao, G., Jiao-Yu, D., Hong-Ping, W., Zhi-Ping, Z., Yan, L., Dong, M., Li-

Rong, S., Xian-En, Z., and Ya-Feng, Z., (2010), Characterization of a monomeric heat-labile

classical alkaline phosphatase from Anabaena sp. PCC7120, Biochemistry (Moscow), 75, 655-

664.

Ninfa, A. J., D. P. Ballou, M. Benore. (2010). Fundamental laboratory approaches for

biochemistry and biotechnology. John Wiley & Sons, Inc.

Voet, Donald, Pratt,Charlotte W., Voet, Judith G. (2012), Principles of Biochemistry. John

Wiley.& Sons, Inc

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