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Jonathan Aivazi

2/18/15
Laboratory 4: Enzyme Kinetics

Objective
The goal of this laboratory is to study the kinetics, specifically the initial velocity
(VO), of the formation of nitrophenol product from pure and crude acid phosphatase
derived from wheat germ extract. This will involve measuring the absorbance of
quenched reactions and plotting the concentrations over a time course, which will enable
the determination of the amount of enzyme that was present in the extract.
Theory
Enzymes are proteins catalysts that increase the speed at which a reaction is
taking place and are present in almost all metabolic and biological processes. Each
enzyme is specific for certain substrates that can bind to it in a tight fitting hold called the
enzyme substrate complex which allows the enzyme to exert its effect on the substrate.
The resulting product is then disengaged from the enzyme, which remains to react with
more substrate.
As catalysts, they increase the rate of reaction by lowering the activation energy
needed to create the desired product. The rate or velocity can be measured by determining
the amount of products formed over time. While the amount of product formed rapidly
increases in the initial minutes of reaction, eventually, the rate slows down until no more
product is formed. Thus the rate, known as the initial velocity (VO), must be calculated
during the initial time frame when rate of product formation is increasing. The initial
velocity is calculated using the following equation:
Equation 1: VO = (nmol product at time 1 nmol product at time 2) / (time 1 time 2)
The initial velocity is of great value as it can be used to yield the amount of
enzyme in the sample. Since the concentration of enzyme is directly proportional to
enzymatic activity, it is possible to determine the amount of enzyme from a preexisting
enzyme amount activity relationship. In practicality, the concentration and kinetics of
the enzyme can be characterized in the laboratory by comparing it to another assay
conducted.

Jonathan Aivazi

2/18/15

Acid phosphatase is an enzyme that is present in wheat germ extract. The germ is
essential for the seed, and is a rich source of proteins, nucleic acids, and enzymes. Acid
phosphatase can be extracted upon grinding and crushing together with a detergent buffer
such as NP40, which lyses the membrane.
The role of acid phosphatase in the fledging plant sesed is to remove phosphate
groups; this can be mimicked in the laboratory by substituting nitrophenol phosphate
instead. Nitrophenol phosphate is an aromtic carbon ring with phosphate, hydroxide, and
nitrite groups. Although nitrophenol phosphate is colorless, the formation of nitrophenol
product is detected by the directly proportional intensity of yellow color, the result upon
the removal of the phosphate by acid phosphatase.
Because the activity of enzymes is dependent on the temperature of the
surroundings, high or low temperature would inhibit the capability of the enzyme by
denaturing it. pH is also another factor in ensuring efficient enzymatic behavior. Acid
phosphatase, as an acid, is optimal at a pH of 4.5. Therefore, this reaction can be
quenched upon addition of KOH, a strong base, to the solution. If the reaction is
quenched at various time points, then the products should be greater as the reaction time
increases.
A separate combination of nitrophenol solutions whose concentrations have been
pre determined could be plotted against absorbance to generate a standard curve. The
linear equation is then applied towards calculating the concentration from the absorbance
values measured at time intervals and belonging to nitrophenol products derived from
both pure and crude acid phosphatase. Once the product concentrations will be plotted
against time, each enzyme source will yield a hyperbolic curve, indicating slightly
different kinetics.
Materials and Reagents

Nitrophenol standards of 0, 25, 50, 100, 200 nmol nitrophenol per 2ml KOH
1 nM Acid phosphatase substrate solution at pH 4.5
50 ug/ml Acid phosphatase
wheat germ
enzyme extraction buffer containing NP 40
1.5 % KOH
Macropipetors

Jonathan Aivazi

2/18/15

Small transfer pipets


16 25 ml glass tubes
Centrifuge and microcentrifuge tubes
Mortar and Pestle
Spectrophotometer

Procedure and Methods


Part A: Preparation of the Enzyme Extract
1. 0.5 g of wheat germ was placed into a mortar and treated with 5 ml of ice cold
extraction buffer
2. The resulting tissue was grinded with the pestle until a homogenous suspension
was formed
3. 1 ml of the solution was transferred to a microcentrifuge tube and spun for 5
minutes
4. The supernatant was then recovered with a pipet and placed in a clean
microcentrifuge tube labeled wheat germ extract
Part B: The Enzyme Assay
1. A rack containing 18 test tubes was obtained and three groups called A,B, & C
were created and consisted of six test tubes labeled 1 6
2. A large pipet was then used to place 1 ml of KOH into each of the test tubes
belonging to the A and B series
3. Two 25 ml beakers were then labeled A and B
4. 10 ml of acid phosphatase substrate was then added to both beaker A beaker B
5. A pipet was then used to trasnfer 1 ml of substrate solution from beaker A to tube
A1 and was repeated for B1
6. 100 l of pure acid phosphatase was added to beaker A and 400 l of wheat germ
extract was applied to beaker B; both beakers were gently stirred
7. At intervals of 0, 2.5, 5, 10, 15, and 20 minutes, 1 ml of solution was removed
from both beaker A and B and placed into the corresponding test tube for that time
frame, followed by absorbance measurement with the spectrophotometer set to
410 nm wavelength
Part C: Measurement of the nitrophenol Standards of the Reaction

Jonathan Aivazi

2/18/15

1. 2 ml of each nitrophenol standard at different concentrations was pippeted in


ascending order in the C series test tubes
2. The absorbance was then determined for each of the test tubes and a standard
curve was generated
3. The absorbance measurements from Part B were then incorporated into the
equation of the standard curve to determine the nmol of nitrophenol product
formed from each of the test tubes; the nmol of each series was then graphed
versus time to reflect the kinetics of crude and pure acid phosphatase
Data and Analysis
This aim of this study was to investigate the kinetics of acid phosphatase as a
catalyst to the production of nitrophenol from nitrophenol phosphate reactant over time.
In order to derive the concentrations of nitrophenol product, the absorbencies of the C
series test tubes were measured, generating a standard curve of known nitrophenol
product concentration. Table 1 summarizes the absorbance data and Graph 1 depicts the
standard curve that was calculated. This graph yielded a reliable R2 value of 0.998,
confirming that the standards worked as well as giving credence to the subsequent
concentrations extrapolated from the y = mx + b equation derived from the graph.
Table 1: Absorbance of C Series nitrophenol is directly proportional to the concentration
Test Tube

Absorbance

Known nmol
Nitrophenol

C1

0.000

C2

0.159

25

C3

0.314

50

C4

0.624

100

C5

1.316

200

C6

2.464

400

Graph 1: Standard Curve of C Series nitrophenol Product

Jonathan Aivazi

2/18/15

Standard curve of Nitrophenol (C - Series)


3.000
2.500

Absorbance (410 nm)

2.000f(x) = 0.01x + 0.01


R = 1
1.500
1.000
0.500
0.000
0 200 400 600
nmol Nitrophenol

The concentrations of the A and B series of acid phosphatase differed in value but
were alike in calculation. The absorbance value of pure or crude from a given time frame
was measured and inserted into the y = mx + b equation above to yield x, which was the
amount of respective nitrophenol product that was formed, in nanomoles. The equation
was rearranged as follows:
Equation 2: (y [Absorbance at time interval] 0.0118) / 0.0062 = x [Concentration]
All the concentrations were determined in this way and graphed against the time in which
they were calculated.
Series A, which contained the pure acid phosphatase and series B, containing
crude acid phosphatase were both plotted on the same graph in order to standardize the
measurements and compare each curve to one another. Table 2 lists the absorbance and
concentration calculated for each series at each time frame of reaction completion. The
kinetics of the acid phosphatase is illustrated in Graph 2.
Time (min)

Test Tube
0

Absorbance

nmoles
Nitrophenol

A1 - Pure

0.000

2.5

A2

0.577

91.1613

A3

0.960

152.9355

10

A4

1.762

282.2903

15

A5

2.608

418.7419

Jonathan Aivazi

2/18/15
20

A6

Time (min)

3.000

Test Tube
0

Absorbance

481.9677
nmoles
Nitrophenol

B1 - Crude

0.000

2.5

B2

1.301

207.9354839

B3

1.726

276.483871

10

B4

2.731

438.5806452

15

B5

3.125

502.1290323

20

B6

3.500

562.6129032

As seen from Graph 2, a slight hyperbolic curve was observed, which indicated
the slowing down of the rate of reaction. This was to be expected since regardless of the
levels of production reached, enzyme kinetics eventually top off and stop as the reaction
refrains from continuing.
Graph 2: Rates of pure and crude acid phosphatase measured at 410 nm

Enzyme Kinetics of Pure (A) and Crude (B) Acid Phosphatase


600

f(x) = 25.87x + 104.95


500 R = 0.89
f(x) = 24.42x + 24.2
400 R = 0.99
B
nmol of Nitophenol product

Linear (B)

300

200

Linear (A)

100
0
0 5 10 15 20 25
Time (min)

Jonathan Aivazi

2/18/15

Analysis of the initial velocities indicated that the initial velocity of the crude was
greater than the pure acid phosphatase. This was calculated using two points at early time
from the curve and inserting them into Equation 1 as follows:
Equation 1: VO = (nmol at time 1 nmol at time 2) / (time 1 time 2)
Pure enzyme = (152.9 91.2) / (5- 2.5) = 24.71
Table 3 lists the VO values for each of the enzyme sources as calculated by the above
equation.
Table 3: VO for crude and pure enzyme
Enzyme source
A - Pure
B - Crude

VO
24.70967742
27.41935484

With respect to the amount of enzyme added to the two beakers, 5 g of purified
acid phosphates was used in the 100 l enzyme sample added to the 10 ml of substrate.
The amount of crude enzyme present in the 400 l of crude sample can be represented in
the following ratio:
5 g Mass of Pure acid phosphatase (A) = Mass of Crude (B) in.5g wheat germ extract
VO (A)
VO (B)
Substituting the values for the denominator derived above yielded a mass value of 5.548
g of enzyme used in 0.5 g of wheat germ extract. Since the amount of enzyme used is
quite similar to the pure amount used it is plausible that we may compare the curves to
each other. To calculate the amount of enzyme in 1 g, we may simply multiply the value
by 2 to a value of 11.097 g.
While there is a high likelihood that our results are reliable, sources of error
occurred that could have played a role in influencing the data outcome. A primary source
of error was that inadequate and inaccurate time keeping persisted due to unfamiliarity
with the protocol, which resulted in reactions occurring either longer or shorter than

Jonathan Aivazi

2/18/15

planned. An additional error was that the solutions of pure and crude may not have been
precisely prepared, potentially impacting results. Another source of error is that the
absorbance readings may not have been so accurate, and although the instrument was
calibrated in between experiments, fluctuations of readings and the handling of dirty
cuvettes could have occurred.
Conclusion
Our results confirm the hypothesis that the kinetics of acid phosphatase can be
characterized and the increased rate of enzyme derived from wheat germ extract as
compared to pure enzyme can be visualized. This difference in rates was quantified by
computing the VO of reaction.
References
1. IND14 Enzyme Kinetics Biochemistry Lab Manual, Department of Biology,
Yeshiva University, New York, 2015.