Beruflich Dokumente
Kultur Dokumente
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
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Jonathan Aivazi
2/18/15
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
Jonathan Aivazi
2/18/15
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
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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
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.