19:59 - 
No comments
No comments
Experiment 3: Experiment on enzyme
TITLE: Experiment On Enzyme
OBJECTIVES:
1.
To
determine the effect of substrate concentration on amylase.
2.
To
determine the effect of different temperature on amylase.
3.
To
determine the effect of different pH on amylase.
INTRODUCTION:
The
study of enzymes is an integral part of the biochemistry curriculum in
universities. Therefore, it is necessary to familiarize the students with the
knowledge of how to develop an enzyme assay and how to observe the effects of
various factors on the activity of an enzyme. In this laboratory, we will
examine the kinetics of α-amylase as found in saliva. Enzyme reacts differently
in different environments. The properties of the environment may influence the
rate of an enzyme to react. For example, pH, temperature or substrate concentration.
In this lab, we will determine the effect of such to amylase.
PROCEDURES:
A)
Preparation
of standard reference
1. Prepare
starch solutions from the stock solution (1.0 mg/ml) into dilutions of 0.01,
0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from the starch stock solution.
2. Iodine
solution is prepared by adding 5 g potassium iodide to 100 ml water. The
dissolved potassium iodide is added with 1 g of iodine and is allowed to
dissolve.
3. Prepare
a standard curve of Absorbance (@ 590 nm) vs. Concentration of a starch/iodine
mixture.
B)
The
effect of substrate concentration
Experiment
of starch hydrolysis in different substrate concentration experiment must be
prepared as the following table.
Data
analysis
1. Calculate starch concentration for each sample after hydrolysis
(SF) through use of the standard curve. The initial starch concentration (S0) is
already known. [S] = (So) – (SF)
2. The velocity (rate of digestion) of the reaction for each
sample can be
calculated as:
V = Δ S/ Δ t = (S0 - SF) / 10 minutes
3. Prepare a table showing rate of hydrolysis (V) at different the
starch
concentrations.
4. Plot a Michaelis-Menten graph.
5. Prepare a graph of 1/starch concentration (x-axis) versus
1/rate of digestion (y-axis). This type of reciprocal graph displaying enzyme kinetics
is a Lineweaver-Burke plot.
6. State the value of Vmax and Michaelis constant Km from your
graph.
C)
The
effect of temperature
Prepare
as the following for the experiment of different temperature.
Data
analysis
Plot the Lineweaver-Burke line for the result of 20, 28, 35 and
40°C.
Compare all three plots.
What are the values of Vmax and Km for all plots?
D)
The
effect of pH
Prepare the following for the experiment using
different pH.
Data
analysis
What are the values of V for all pH?
Compare the velocity for each of the pH test
RESULTS:
A)
Preparation
of standard reference
Graph 1:
Standard Curve
A)
The
effect of substrate concentration
Test tube
|
So
(mg/ml)
|
Absorbance
(nm)
|
SF
(mg/ml)
|
S = (So - SF)
|
1/S
|
V =
S/10 minutes
|
1/V
|
1
|
0
|
0.018
|
0.005
|
-0.005
|
-200
|
-0.0005
|
-2000
|
2
|
0.01
|
0.06
|
0.01
|
0
|
0
|
0
|
0
|
3
|
0.025
|
0.148
|
0.03
|
0
|
0
|
0
|
0
|
4
|
0.05
|
0.279
|
0.07
|
-0.02
|
-50
|
-0.002
|
-500
|
5
|
0.1
|
0.472
|
0.12
|
-0.02
|
-50
|
-0.002
|
-500
|
6
|
0.3
|
1.245
|
0.3
|
0
|
0
|
0
|
0
|
7
|
0.5
|
1.912
|
0.49
|
0.01
|
100
|
-0.001
|
1000
|
8
|
0.7
|
2.471
|
0.61
|
0.09
|
11.1
|
-0.009
|
111.11
|
9
|
1
|
3.324
|
0.81
|
0.19
|
5.26
|
0.019
|
52.6
|
Graph 2:
Michaelis-Menten Graph
Graph 3:
Lineweaver-Burke Graph
B) The
Effect of Different Temperature
Temperature: 10ºC
Test
Tube
|
So
(mg/ml)
|
Absorbance
(nm)
|
Sf
(mg/ml)
|
S
=
(So
– Sf)
|
1/S
|
V
= S/20mins
|
1/V
|
1
|
0.000
|
0.298
|
0.0275
|
-0.0275
|
-36.364
|
-0.001375
|
-727.27
|
2
|
0.010
|
0.201
|
0.0384
|
-0.0284
|
-35.211
|
-0.00142
|
-704.22
|
3
|
0.025
|
0.204
|
0.0391
|
-0.0141
|
-70.922
|
-0.000705
|
-1418.44
|
4
|
0.050
|
0.190
|
0.0361
|
0.0139
|
71.94
|
0.000695
|
1438.85
|
5
|
0.100
|
0.213
|
0.0407
|
0.0593
|
16.863
|
0.002965
|
337.268
|
6
|
0.300
|
0.172
|
0.0325
|
0.2675
|
3.7383
|
0.01337
|
74.766
|
7
|
0.500
|
0.173
|
0.0326
|
0.4674
|
2.1395
|
0.02337
|
42.7899
|
8
|
0.700
|
0.204
|
0.0391
|
0.6609
|
1.51308
|
0.03304
|
30.2618
|
9
|
1.000
|
0.253
|
0.0491
|
0.9509
|
1.0516
|
0.0475
|
21.0327
|
Temperature: 28ºC
Test
Tube
|
So
(mg/ml)
|
Absorbance
(nm)
|
Sf
(mg/ml)
|
S
=
(So
– Sf)
|
1/S
|
V
= S/20mins
|
1/V
|
1
|
0.000
|
0.185
|
0.0350
|
-0.035
|
-28.5
|
-0.00175
|
-571.43
|
2
|
0.010
|
0.162
|
0.0304
|
-0.0204
|
-49.02
|
-0.00102
|
-980.39
|
3
|
0.025
|
0.154
|
0.0286
|
-0.0036
|
-277.78
|
-0.00018
|
-5555.56
|
4
|
0.050
|
0.240
|
0.0465
|
0.0035
|
285.71
|
0.000175
|
5714.28
|
5
|
0.100
|
0.131
|
0.0239
|
0.0761
|
13.1406
|
0.003805
|
262.81
|
6
|
0.300
|
0.190
|
0.0361
|
0.2639
|
3.7893
|
0.01319
|
75.7862
|
7
|
0.500
|
0.209
|
0.0401
|
0.4599
|
2.1744
|
0.00153
|
653.595
|
8
|
0.700
|
0.163
|
0.0306
|
0.6694
|
1,4939
|
0.03347
|
29.877
|
9
|
1.000
|
0.190
|
0.0361
|
0.9639
|
1.03745
|
0.04819
|
20.7490
|
Temperature: 35ºC
Test
Tube
|
So
(mg/ml)
|
Absorbance
(nm)
|
Sf
(mg/ml)
|
S
=
(So
– Sf)
|
1/S
|
V
= S/20mins
|
1/V
|
1
|
0.000
|
0.245
|
0.0475
|
-0.0475
|
-21.0526
|
-0,002375
|
-421.05
|
2
|
0.010
|
0.144
|
0.0267
|
-0.0167
|
-59.8802
|
-0.000835
|
-1197.606
|
3
|
0.025
|
0.143
|
0.0265
|
-0.0015
|
-666.67
|
0.00007
|
-1333.3
|
4
|
0.050
|
0.141
|
0.0260
|
0.024
|
41.667
|
0.003
|
1000
|
5
|
0.100
|
0.152
|
0.0283
|
0.0717
|
13.947
|
0.000855
|
1169.591
|
6
|
0.300
|
0.192
|
0.0365
|
0.2635
|
3.79506
|
0.013175
|
75.90
|
7
|
0.500
|
0.839
|
0.0675
|
0.4325
|
2.3121
|
0.02162
|
46.2428
|
8
|
0.700
|
0.161
|
0.0303
|
0.6697
|
1.4932
|
0.03349
|
29.6864
|
9
|
1.000
|
0.106
|
0.0177
|
0.9823
|
1.01802
|
0.04911
|
20.3604
|
Temperature: 40ºC
Test
Tube
|
So
(mg/ml)
|
Absorbance
(nm)
|
Sf
(mg/ml)
|
S
=
(So
– Sf)
|
1/S
|
V
= S/20mins
|
1/V
|
1
|
0.000
|
0.082
|
0.014
|
-0.014
|
-71.4285
|
-0.0007
|
1428.57
|
2
|
0.010
|
0.107
|
0.019
|
-0.009
|
-111.111
|
0.00045
|
2222.22
|
3
|
0.025
|
0.110
|
0.021
|
0.004
|
250
|
0.0002
|
5000
|
4
|
0.050
|
0.093
|
0.016
|
0.034
|
29.412
|
0.0017
|
588.24
|
5
|
0.100
|
0.100
|
0.018
|
0.082
|
12.195
|
0.0009
|
1111.11
|
6
|
0.300
|
0.088
|
0.015
|
0.285
|
3.5088
|
0.00075
|
1333.33
|
7
|
0.500
|
0.091
|
0.015
|
0.485
|
2.06185
|
0.00075
|
1333.33
|
8
|
0.700
|
0.074
|
0.012
|
0.688
|
1.4535
|
0.0006
|
1666.66
|
9
|
1.000
|
0.184
|
0.037
|
0.963
|
1.03842
|
0.04815
|
20.768
|
Graph 4: Lineweaver-Burke
Temperature 10°C (Vmax and KM)
Vmax= 32
Km= -5.80
Vmax= 32
Km= -5.80
Temperature 28°C (Vmax and KM)
Vmax = 32
Km= -2.60
Vmax = 32
Km= -2.60
Temperature 35°C (Vmax and KM)
Vmax = 32
Km= -7.65
Vmax = 32
Km= -7.65
Temperature 40°C (Vmax and KM)
Vmax = 32
Km = -1.00
Vmax = 32
Km = -1.00
C) The
effect of Different pH value
Test tube
|
pH
|
Absorbance
|
So
|
Sf
|
S=( So - Sf )
|
V=s/time
|
1
|
4
|
0.840
|
0.5
|
0.200
|
0.300
|
0.015
|
2
|
5
|
0.432
|
0.5
|
0.100
|
0.400
|
0.020
|
3
|
6
|
0.247
|
0.059
|
0.441
|
0.022
|
|
4
|
7
|
0.208
|
0.5
|
0.045
|
0.455
|
0.023
|
5
|
8
|
0.564
|
0.5
|
0.131
|
0.369
|
0.018
|
6
|
9
|
0.108
|
0.5
|
0.021
|
0.479
|
0.023
|
7
|
10
|
0.061
|
0.5
|
0.020
|
0.480
|
0.024
|
blank
|
water
|
0.252
|
0.5
|
0.059
|
0.441
|
0.022
|
DISCUSSION:
The Effect of Substrate Concentration
Catalysts are agents that speed up chemical processes. Almost all catalysts used by living cells are
called enzymes. Enzymes are proteins and
each cell produces hundreds of them.
Enzymes accelerate the velocity of virtually all reactions that occur in
biological systems including those involved in breakdown, synthesis and
chemical transfers.
The
binding of an enzyme to its substrate is an essential part of the
enzyme-catalyzed reaction. As the
Lineweaver-Burke graph shown above, at low substrate concentration, the active
site on the enzyme is not saturated by substrate and thus the enzyme is not
working at full capacity. As the
concentration of the substrate increases, the sites are bound to a greater
degree until saturation when no more sites are available for substrate
binding. At this saturating substrate
concentration, the enzyme is working at full capacity and the maximum velocity
(Vmax) of the reaction is seen as 0.019 and the Km is 0.03. The Km of substrate would indicate that the substrate-binding site would be half saturated when the substrate is present at that concentration.
The
consequence of saturating the enzyme with substrate on the reaction rate is
shown in Michaelis-Menten graph. The initial velocity of the
reaction increases in a hyperbolic manner as the substrate concentration is
increased. The increase in reaction rate
is proportional to the concentration of the enzyme-substrate complex. Thus, Vmax occurs because the
enzyme becomes saturated with substrate.
The
Effect of Different Temperature oon Enzyme reactions.
Commonly
we know that enzymes reaction is mainly influence by the temperature that
surrounds the reaction this happen because certain factors that focusing on the
molecular level of reactions between the atoms within the enzymes.As the
temperature increasing the reaction by increasing the collisions as the atoms
receive energy so it will collide often and this also cause the lowering of
activation energy dor exergonic reactions.Next,what happen is that the number
of collision increase tremendously because in order for the reaction between
enzymes and substares to occur they need to collide so that it could bind to
the active site.Lastly is that when the temperature is too high the temperature
inside the molecule is going to become higher as there are certain kinetic
energy contributing to the increase of temperature inside the molecules causing
the bonds becomes weak and the enzymes proteins begin to become denatured.During
this stage the reaction is no longer increasing but it begin to slow down and
eventually stops.
That is
what suppose to happen and from this experiment we are going to measure the
velocity of the reaction in order to
know the rate of reaction influence by the temperature.There are 4 different
temperatures which is 10°C.28°C.35°C and
40° C.Based on the results mostall the grapg being plotted using the Lineweaver
Burke graph but none showed a good patterns as there are no saturated part as
the graph is happen to be in a straight line.Actually if the grapg is good we
are able to know the Vmax and the Km from the grapg itself but based on the grapg
we obtained all showed negative resulta because all the reading is zero thus we
are unable to really compare the reaction rate based on the velocity for the
reaction of enzyme from different temperature.
The
effect of Different pH values
The
activity of enzymes is also greatly influenced by acidity or alkalinity. Excess acidity or alkalinity generally causes
denaturation and inactivation of enzymes. Enzymes have an optimum pH or pH
range in which their activity is maximum. At higher or lower pH their activity
decreases. The active sites on enzymes are often composed of ionizable groups
which must be in the appropriate ionic form to maintain the conformation of the
active site, bind the substrates or catalyze the reaction. On the other hand
one or more of the substrates may contain ionizable groups and only one ionic
form of that substrate may bind to the enzyme or undergo catalysis.
CONCLUSION:
In conclusions,substrate which is higher
in reaction rate when substrate concentration is high and when the temperature
is rising but both has the satirated point where the reaction could not go any
further and eventually stops.As for the pH when the excess acidity or
alkalinity actually cause denaturation and inactivation of enzymes so enzymes
works best only on certain specific temperature.
REFERENCE:
Enzyme
Kinetics (2012). Retrieved on May 2,
2013 from
http://www.columbia.edu/itc/chemistry/chem-c2407/hw/ENZYME_KINETICS.pdf
A look into enzyme kinetics:
Some introductory experiments (2010).
Retrived on 2 MAy, 2013 from
http://onlinelibrary.wiley.com/doi/10.1016/0307-4412(92)90121-2/pdf
Effects Of Temperature On Enzyme Activity retrieved on May 2,2013 from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/enz_act.htm
0 comments:
Post a Comment