Upload
adnan-malak
View
441
Download
0
Embed Size (px)
Citation preview
H83 BCE LAB REPORT
NAME: Adnaan Malak
Student id: 012117
Group no: 7
Date of experiment: 30/03/2015
Date of submission: 06/04/2015
Lecturer: Dr. Lau Phei Li
SUMMARY: The following report is based on an experiment that was conducted to study how
enzyme catalysed reactions behave in different conditions. In this experiment
lipase was used as the enzyme that catalysed the hydrolysis of tributyrin into
butyric acid and glycerol. The Michaelis-Menten kinetics model was used as a
reference to study the effect of reaction rate on the concentration of substrate
and enzyme. The details on MM kinetics are further discussed in the report and
Hanes model is selected as a perfect model to calculate value of Kcat. Trend for
effect of change in substrate concentration and change in enzyme concentration
on the initial rate of reaction is also discussed in the discussion section of the
report.
INTRODUCTION: Enzymes are effective biological catalysts. They speed up the rate of reaction by
providing alternative route with lower activation energy. One of the classic
examples is the digestion /breakdown of fats found in the food to fatty acids in
the stomach by the enzyme lipase. Normally this reaction would take a long time
to occur and the food containing the necessary nutrients would just exit the body
but due to presence of lipase it is digested much faster. Enzymes are very
selective; they only bind with molecules which have desired active sites. Enzymes
get affected mostly by change in temperature or pH.
A study conducted has shown that enzymes generally follow the michaelis-
menton kinetics when they act as catalyst in the reactions. A much more detailed
abstract about the michaelis-menton was already discussed in the lab manual. In
our experiment, the enzyme tested is lipase and the substrate is tributyrin. Lipase
is mainly produced in the pancreas. The stomach also produces small amounts of
enzyme lipase. This enzyme catalyzes the hydrolysis of ingested food fat in the
body to fatty acids and glycerol so that it can be absorbed in the intestines.
Tributyrin is a triglyceride naturally found in foods such as butter and can be
described as a liquid fat with an acrid taste. The tributyic acid produced is
neutralized by NaOH to find the rate of reaction. The experiment was run until 5
minutes and a volume reading of NaOH and pH of the solution was noted every
30 seconds.
The main objectives of these experiments are:
• To determine the effect of the substrate concentration on the initial
rate of reaction for a constant concentration of enzyme.
• To determine Vmax and Km of the Michaelis-Menten kinetics.
• To determine the effect of enzyme concentration on the initial rate
of reaction.
• To determine Kcat
The hydrolysis of tributyrin to produce fatty acids and glycerol is assumed
to follow the enzymatic kinetics of the Michaelis-Menten model. This
model was developed in 1913. It provided a theoretical explanation for the
reaction rate using hypothesized reaction mechanism. It assumes that the
enzyme, E and substrate, S combine to form a complex ES, which then
dissociates into product P and free enzyme E.
S+E ↔ES→P+E
The Michaelis-Menten model is a pseudo steady state enzyme
kinetic. From this plot, it is difficult to determine the values of Km and
Vmax. So to determine the Vmax and the Km values, Lineweaver-Burk,
Eadie Hofstee and Hanes models were used. These models fairly give us a
good representation of Michaelis-Menten kinetics in a much more easy to
read plots, so that Vmax and Km values can be determined.
Lineweaver-Burk model:
[ ]
Y-axis is 1/Vo, Xaxis is 1/So
Slope equals to Km/Vm
Y-intercept equals to 1/Vm
Eadie Hofstee model:
Y-axis is Vo and x-axis is Vo/So
Slope equals to –Km
Y-intercept equals to Vmax
Hanes model:
Y-axis is So/Vo and x-axis is So
Slope equals to 1/Vmax
Y-intercept equals to Km/Vmax
RESULTS: EXPERIMENT 1: SUBSTRATE A
Volume of enzyme: 0.5ml
Table 1: Results of using 0.5ml enzyme and substrate A
Volume of enzyme:1.0ml
Table 2: Results of using 1.0ml enzyme and substrate A
Time(min) pH Volume of NaOH added(ml)
0 6.81 2.064
0.5 6.81 0.093
1 6.99 0.146
1.5 7.02 0.170
2 7.02 0.214
2.5 7.02 0.214
3 7.02 0.234
3.5 7.02 0.254
4 7.02 0.274
4.5 6.99 0.292
5 7.01 0.304
Time pH Volume of NaOH added
0 6.83 2.035
0.5 6.52 0.186
1 6.82 0.286
1.5 7.01 0.342
2 6.99 0.378
Volume of enzyme:1.5ml
Table 3: Results of using 1.5ml enzyme and substrate A
Time pH Volume of NaOH added
0 6.82 2.042
0.5 6.6 0.096
1 6.8 0.176
1.5 7.01 0.234
2 7.01 0.260
2.5 7.00 0.280
3 7.01 0.290
3.5 7.01 0.310
4 7.01 0.324
4.5 7.01 0.340
5 7.02 0.356
2.5 6.99 0.422
3 7.03 0.446
3.5 7.01 0.460
4 6.99 0.478
4.5 7.03 0.498
5 7.02 0.510
Figure 1: Plot of volume of NaOH vs Time for substrate A
EXPERIMENT 2: SUBSTRATE B
Volume of enzyme:0.5mL
Table 4: Results of using 0.5ml enzyme and substrate B
Time pH Volume of NaOH added
0 6.81 3.322
0.5 6.47 0.160
1 6.62 0.286
1.5 6.72 0.388
2 6.76 0.478
2.5 6.82 0.564
3 6.87 0.644
3.5 6.91 0.722
4 6.97 0.798
4.5 6.99 0.860
5 7.01 0.918
y = 0.0529x + 0.0655
y = 0.0851x + 0.1515
y = 0.0615x + 0.0886
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 1 2 3 4 5 6
Vo
lum
e o
f N
aOH
ad
ded
(ml)
Time(min)
0.5 ml of enzyme
1 ml of enzyme
1.5 ml of enzyme
Linear (0.5 ml of enzyme )
Linear (1 ml of enzyme)
Linear (1.5 ml of enzyme )
Volume of enzyme: 1.0ml
Table 5: Results of using 1.0ml enzyme and substrate B
Time pH Volume of NaOH added
0 6.89 2.874
0.5 6.41 0.224
1 6.61 0.292
1.5 6..77 0.378
2 6.92 0.456
2.5 7.01 0.504
3 7.01 0.540
3.5 7.01 0.574
4 7.0 0.606
4.5 7.0 0.632
5 7.01 0.658
Volume of enzyme:1.5ml
Table 6: Results of using 1.5ml enzyme and substrate C
Time pH Volume of NaOH added
0 6.81 2.806
0.5 6.54 0.158
1 6.66 0.254
1.5 6.78 0.342
2 6.9 0.422
2.5 7 0.480
3 7 0.518
3.5 7 0.550
4 7.01 0.592
4.5 7 0.630
5 7.01 0.660
Figure 2: Plot of volume of NaOH vs Time for substrate B
EXPERIMENT 3: SUBSTRATE C
Volume of enzyme:0.5ml
Table 7: Results of using 0.5ml enzyme and substrate C
Time pH Volume of NaOH added
0 6.81 5.532
0.5 6.75 0.098
1 6.99 0.160
1.5 7.01 0.166
2 7.01 0.176
2.5 7.03 0.188
3 7.0 0.188
3.5 7.02 0.2
4 7.03 0.212
4.5 7.01 0.212
5 7.02 0.222
y = 0.1775x + 0.0853
y = 0.1153x + 0.154 y = 0.1221x + 0.1135 y = 0.1221x + 0.1135 y = 0.1221x + 0.1135 y = 0.1221x + 0.1135
0
0.2
0.4
0.6
0.8
1
1.2
0 1 2 3 4 5 6
Vo
lme
of
NaO
H a
dd
ed(m
l)
Time (min)
0.5 ml of enzyme
1 ml of enzyme
1.5 ml of enzyme
Linear (0.5 ml of enzyme )
Linear (1 ml of enzyme )
Linear (1.5 ml of enzyme )
Linear (1.5 ml of enzyme )
Linear (1.5 ml of enzyme )
Linear (1.5 ml of enzyme )
Volume of enzyme:1.0ml
Table 8: Results of using 1.0ml enzyme and substrate C
Time pH Volume of NaOH added
0 6.81 5.498
0.5 6.55 0.128
1 6.77 0.218
1.5 6.97 0.290
2 7.01 0.310
2.5 7.01 0.320
3 7.00 0.328
3.5 7.00 0.336
4 7.00 0.346
4.5 7.01 0.354
5 7.01 0.366
Volume of enzyme:1.5ml
Table 9: Results of using 1.5ml enzyme and substrate C
Time pH Volume of NaOH added
0 6.8 5.37
0.5 6.68 0.126
1 6.94 0.208
1.5 7.02 0.226
2 7.03 0.236
2.5 7.00 0.233
3 7.01 0.246
3.5 7.03 0.258
4 7.01 0.258
4.5 7.02 0.268
5 7.03 0.278
Figure 3: Plot of NaOH vs Time for substrate C
EXPERIMENTAL VALUES FOR INTIAL RATE OF REACTION WITH VARIABLE
ENZYME CONCENTRATION:
Table 10 : Results of initial rate of reaction with variable enzyme concentration
Substrate A
So( mol/ml) Enzyme (LU/L) initial rate( mol/min)
1.14E-05 2.44E-02 2.65E-06
1.14E-05 4.76E-02 4.26E-06
1.14E-05 6.98E-02 3.08E-06
Substrate B
So( mol/ml) Enzyme (LU/L) initial rate( mol/min)
2.28E-05 2.44E-02 8.88E-06
2.28E-05 4.76E-02 5.68E-06
2.28E-05 6.98E-02 6.11E-06
y = 0.0328x + 0.0837
y = 0.0587x + 0.1256
y = 0.0397x + 0.1137
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 1 2 3 4 5 6
Vo
lum
e o
f N
aOH
ad
ded
Time (min)
0.5 ml of enzyme
1 ml of enzyme
1.5 ml of enzyme
Linear (1 ml of enzyme )
Linear (1.5 ml of enzyme )
Linear (1.5 ml of enzyme )
Substrate C
So( mol/ml) Enzyme (LU/L) initial rate( mol/min)
3.41E-05 2.44E-02 1.64E-06
3.41E-05 4.76E-02 2.94E-06
3.41E-05 6.98E-02 1.99E-06
Figure 4: Plot of enzyme concentration vs initial rate
LINEWEAVER-BURK MODEL DATA POINTS AND PLOT:
Table 11: Data for LINEWEAVER-BURK MODEL
0.00E+00
2.00E-06
4.00E-06
6.00E-06
8.00E-06
1.00E-05
0.00E+001.00E-022.00E-023.00E-024.00E-025.00E-026.00E-027.00E-028.00E-02
init
ial r
ate
(mo
l/m
in)
Enzyme concentration(LU/L)
Substrate A
Substrate B
Substrate C
LB Plot
0.5 ml v0 So 1/vo 1/So
A 2.65E-06 1.14E-05 3.78E+05 8.79E+04
B 8.88E-06 2.28E-05 1.13E+05 4.39E+04
C 1.64E-06 3.41E-05 6.10E+05 2.93E+04
1 ml v0 So 1/vo 1/So
A 4.3E-06 1.1E-05 2.4E+05 8.8E+04
B 5.7E-06 2.3E-05 1.8E+05 4.4E+04
C 2.9E-06 3.4E-05 3.4E+05 2.9E+04
Figure 5: Plot for LINEWEAVER-BURK model
Table 12: Values of Km and Vmax for LINEWEAVER-BURK MODEL
enzyme volume (ml) Km(mol/ml) Vmax(mol/min) Regression(R2) 0.5 -3.6E-06 2.2E-06 0.041
1 -3.6E-06 2.4E-06 0.072
1.5 -3.5E-06 3.2E-06 0.1554
y = -1.6478x + 455347
y = -1.0788x + 308592
y = -1.4967x + 411322
0.00E+00
1.00E+05
2.00E+05
3.00E+05
4.00E+05
5.00E+05
6.00E+05
7.00E+05
0.00E+00 2.00E+04 4.00E+04 6.00E+04 8.00E+04 1.00E+05
1/V
o(m
in/m
ol)
1/So(ml/mol)
0.5 ml enzyme concentration
1 ml enzyme concentration
1.5 ml concentration
Linear (0.5 ml enzymeconcentration )
Linear (1 ml enzymeconcentration )
Linear (1.5 ml concentration )
1.5 ml v0 So 1/vo 1/So
A 3.1E-06 1.1E-05 3.3E+05 8.8E+04
B 6.1E-06 2.3E-05 1.6E+05 4.4E+04
C 2.0E-06 3.4E-05 5.0E+05 2.9E+04
EDDIE-HOFSTEE MODEL DATA POINTS AND PLOT:
Table 13: Table for EDDIE-HOFTSEE MODEL
Figure 6 : Plot for EDDIE-HOFSTEE model
y = 2E-05x - 2E-07
y = 5E-06x + 3E-06 y = 1E-05x + 1E-06
0.00E+00
1.00E-06
2.00E-06
3.00E-06
4.00E-06
5.00E-06
6.00E-06
7.00E-06
8.00E-06
9.00E-06
1.00E-05
0.00E+00 1.00E-01 2.00E-01 3.00E-01 4.00E-01 5.00E-01
Vo
(mo
l/m
l)
Vo/So(ml/min)
0.5 ml enzyme concentration
1 ml enzyme concentration
1.5 ml enzyme concentration
Linear (0.5 ml enzymeconcentration )
Linear (1 ml enzymeconcentration )
Linear (1.5 ml enzymeconcentration )
Eadie Hofstee Plot
0.5 ml v0 So vo/[so]
A 2.65E-06 1.14E-05 2.32E-01
B 8.88E-06 2.28E-05 3.90E-01
C 1.64E-06 3.41E-05 4.80E-02
1 ml v0 So vo/[so]
A 4.26E-06 1.14E-05 3.74E-01
B 5.68E-06 2.28E-05 2.49E-01
C 2.94E-06 3.41E-05 8.60E-02
1.5 ml v0 So vo/[so]
A 3.08E-06 1.14E-05 2.70E-01
B 6.11E-06 2.28E-05 2.68E-01
C 1.99E-06 3.41E-05 5.82E-02
Table 14: Value of Km and Vmax for EDDIE-HOFSTEE model
HANES MODEL DATA POINTS AND PLOT:
Table 15 : Data for HANES model
3- Hanes plot
0.5 ml v0 So S0/vo
A 0.000002645 1.13772E-05 4.301386122
B 0.000008875 2.27543E-05 2.56386846
C 0.00000164 3.41315E-05 20.81188956
1 ml v0 So
A 0.000004255 1.13772E-05 2.673834616
B 0.000005675 2.27543E-05 4.009574024
C 0.000002935 3.41315E-05 11.62913079
1.5 ml v0 So
A 0.000003075 1.13772E-05 3.699891477
B 0.000006105 2.27543E-05 3.727163404
C 0.000001985 3.41315E-05 17.19470976
Table 16 : Values of Km and Vmax for HANES plot
enzyme concentration (ml) Km(mol/ml) Vmax (mol/min) Regression(R2)
0.5 -2.0E-05 -2.0E-07 0.8183
1 -5.0E-06 3.0E-06 0.3005
1.5 -1.0E-05 1.0E-06 0.4882
enzyme concentration km Vmax Regression
0.5 -1.00E-05 1.38E-06 0.6719
1 -7.24E-06 2.54E-06 0.7515
1.5 -8.92E-06 1.69E-06 0.859
Figure 7: Plot for HANES model
EFFECT OF VARIABLE SUBSTRATE CONCENTRATION ON THE INITIAL RATE
OF REACTION:
Table 17: Variable substrate concentration vs initial rate
y = 725598x - 7.2848
y = 393564x - 2.8511
y = 593066x - 5.2876
0
5
10
15
20
25
0 0.00001 0.00002 0.00003 0.00004
So/V
o
So
0.5 ml enzyme concentration
1ml enzyme concentration
1.5 ml enzyme concentration
Linear (0.5 ml enzymeconcentration )
Linear (0.5 ml enzymeconcentration )
Linear (1ml enzymeconcentration )
enzyme Concentration 0.024390244 0.04761905 0.069767442
Substrate concentration initial rate initial rate initial rate A 1.13772E-05 2.65E-06 4.26E-06 3.08E-06
B 2.27543E-05 8.88E-06 5.68E-06 6.11E-06
C 3.41315E-05 1.64E-06 2.94E-06 1.99E-06
Figure 8 : Plot for Variable substrate concentration vs initial rate
Table 18: Kcat values from HANES model
Enzyme Volume (ml) Kcat (mol/min)
0.5 0.00380
1.0 0.00358
1.5 0.00351
0.00E+00
2.00E-06
4.00E-06
6.00E-06
8.00E-06
1.00E-05
0 0.00001 0.00002 0.00003 0.00004
Init
ial R
ate
(mo
l/m
in)
Substrate Concentration (mol/ml)
enzyme concentration of0.02439
enzyme concentration of 0.048
enzyme concentration of0.0698
DISCUSSION:
EFFECT OF SUBSTRATE CONCENTRATION ON INITIAL RATE FOR CONSTANT
ENZYME CONCENTRATION
In theory more the substrate more the reaction rate should increase. This is
because more active sites are available a low substrate concentration. This
trend is clearly shown in Figure 8 .Whereas the substrate concentration
increases the reaction rate increases until it reaches a maximum point. In
our case for enzyme concentration of 0.02349,0.04761 and 0.06976 the
maximum values obtained for reaction rate are: 8.88E-06,6.11E-06 and
5.86E-06. When it reaches its maximum value then there is a drop in the
reaction rate as the enzyme gets saturated and thus there is no available
active sites for the substrate to bind with the enzyme.
According to (Jonathan crow, 2010) when the concentration of substrate
increases to a very high level, the substrate molecule competes for the
same active site on the enzyme to bind on. It can also be that the second
substrate binds on the specialized sites of enzymes where it can act as
allosteric inhibitor (Worthington-biochem). Hence due to these reason a
decrease in the reaction rate is observed .
DETERMING Vm and Kmax VALUE ASSUMING MICHAELIS-MENTEN
KINETICS
Values of Km and Vmax are determined by the three models: Lineweaver-
Burk, Eadie Hofstee and Hanes. Tables 12,14 and 16 displays the Km and
Vmax values. There are some limitations on using the three modified
models instead of the Michaelis-Menten original model. For Lineweaver-
Burk plot the linearity of LB plot is much less linear than the other two
plots; as also shown in figure 5,6 and 7 the R2 value for the LB plot is less
than the other two plots. There are several limitations associated with the
LB plot. These include:
• Difficult to determine how much graph paper is needed for the plot
to reach the x-axis.
• It allows low concentration of substrate to be used which can skew
the plot.
• Deviations from linearity are less easy to spot compared with other
means of interpretation.
For Eadie-Hofstee plot, the uneven spacing and the large values of inverse for
lower substrate concentration is overcome. But since Vo is used in both the axis
for plotting the graph the errors in measuring the Vmax are multiplied
considerably. From regression analysis shown in table 14 and 16 as compared to
Hanes plot has a lower R2 value which by no doubt makes Hanes plot the best
model to refers.
For hanes plot, the problem of uneven spacing and high values at low substrate
concentration is overcome by multiplying the term So to both the x-axis and the
y-axis (refer to introduction section to see the equation). But the disadvantage of
this model is that since So is use in both the axis. Due to which the drawback
results in an error value for true concentrations of but the overall error for the Km
and Vmax values are generally acceptable.
EFFECT OF ENZYME CONCENTRATION ON INITIAL RATE OF REACTION FOR
CONSTANT ENZYME CONCENTRATION
As it is observed from the figure 4 ; an increase in enzyme concentrations
leads to an increase in the rate of reactions for only substrate A and C . The
reason for substrate B not showing the similar trend can be because of the
systematic error or random error performed by Group number 22 from
which the values for 1.0 and 1.5 ml of enzyme added was taken. As for
substrate A and C the values for 0.5 and 1 ml were taken by my group
which shows the correct trend. This trend can be justified by the fact that
as the concentration of enzymes increases the availability of active sites
also increases which results in more substrates binding with the enzyme
resulting in a high rate of reaction. The initial rate of reaction h substrate A
is higher than C which could be because of substrate inhibition as explained
earlier.
Determining Kcat value for the enzyme
The Hanes plot is selected to determine the Kcat value because of the best
linear line obtained and has the largest R2 values.
How closely does the reaction comply with Michaelis-Menten kinetics?
Figure : ideal graph for MM equation
Figure: experimental graph obtained for MM equation
As it can be seen from the above two graphs for the following equation:
For the following experiment the reaction doesn’t completely comply with MM
equation. As it can also be seen from table 16 that the values obtained for Km are
in negative which suggests the involvement of experimental errors in our
experiments. But as said above it doesn’t completely comply which means that
the MM kinetics predicts that the Km and Kcat values do not change with the
increasing enzyme concentration. This trend is correctly followed by the Kcat
values that have been determined. But if we ignore the negative values then it
can be safely assumed that the reaction complies with the Michaelis-menten
Kinetics.
0.00E+00
2.00E-06
4.00E-06
6.00E-06
8.00E-06
1.00E-05
1.20E-05
1.40E-05
0 0.00001 0.00002 0.00003 0.00004
Vo
So
0.5 ml enzyme
1 ml enzyme
1.5 ml
CONCLUSION: The 5 criteria discussed in discussion part concludes the following:
The rate of reaction increases with increasing substrate concentration until
the enzyme active sites get saturated.
The rate of reaction increases with increasing enzyme concentration until
there is no more available substrate to be converted to products. Only
exception is for substrate B where experimental errors are involved.
The Vmax and Km values for all the LB, EH and Hanes model are
determined but only those from the Hanes model are selected.
The value of Kcat is determined using the Hanes model.
The reaction is not in perfect compliance to the Michaelis-Menten kinetic
model. However when the graph is plotted using the Hanes model, the
values of Vmax and Kcat can be determined to sufficient accuracy because
the lines obtained can be approximated to be linear with quite low errors.
REFERENCE:
1. John R. Holum, 1995. Elements of General, Organic and Biological Chemistry,
9th Edition. 9 Edition. Wiley.
2. Anon, (2015). [online] Available at: Mpbio.com, (2015). TRIBUTYRIN
(02103111) - MP Biomedicals. [online] Available at:
http://www.mpbio.com/product.php?pid=02103111&country=129 [Accessed
3 Mar. 2015].
3. Worthington-biochem.com, (2015). Enzyme Concentration (Introduction to
Enzymes). [online] Available at: http://www.worthington-
biochem.com/introbiochem/enzymeconc.html [Accessed 4 Mar. 2015].
4. Ucl.ac.uk, (2015). Untitled Document. [online] Available at:
http://www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm [Accessed 27 Feb.
2015].
5. Worthington-biochem.com, (2015). Enzyme Concentration (Introduction to
Enzymes). [online] Available at: http://www.worthington-
biochem.com/introbiochem/enzymeconc.html [Accessed 1 Mar. 2015].
6. Dr Edward Group III DC, D. (2011). The Health Benefits of Lipase. [online]
Dr. Group's Natural Health & Organic Living Blog. Available at:
http://www.globalhealingcenter.com/natural-health/lipase/ [Accessed 3 Mar.
2015].
7. Abraham Mazur, 1971. Textbook of Biochemistry. 10th Revised edition
Edition. Saunders (W.B.) Co Ltd.
8. Enzymeessentials.com, (2015). Supplementing with Digestive Enzymes.
Lipase-Digestive enzyme to digest fats and lipids. [online] Available at:
http://www.enzymeessentials.com/HTML/lipase.html [Accessed 4 Mar.
2015].
9. Gaschott, T., Steinhilber, D., Milovic, V. and Stein, J. (2001). Tributyrin, a
Stable and Rapidly Absorbed Prodrug of Butyric Acid, Enhances
Antiproliferative Effects of Dihydroxycholecalciferol in Human Colon Cancer
Cells. The Journal of Nutrition, [online] 131(6), pp.1839-1843. Available at:
http://jn.nutrition.org/content/131/6/1839.full [Accessed 2 Mar. 2015].
10.Mpbio.com, (2015). TRIBUTYRIN (02103111) - MP Biomedicals. [online]
Available at:
http://www.mpbio.com/product.php?pid=02103111&country=129 [Accessed
4 Mar. 2015].
11.University of Maryland Medical Center, (2015). Lipase. [online] Available at:
http://umm.edu/health/medical/altmed/supplement/lipase [Accessed 6 Mar.
2015].
12.Athel Cornish-Bowden, 2012. Fundamentals of Enzyme Kinetics. 4 Edition.
Wiley-Blackwell.
13.2015. Untitled Document. [ONLINE] Available
at:http://www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm. [Accessed 20
March 2015].
14.Jonathan Crowe, 2010. Chemistry for the Biosciences: The Essential
Concepts. 2 Edition. Oxford University Press.
APPENDIX:
SUBSTRATE CONCENTRATION
From Perry’s Chemical Handbook:
Density of Tributyrin, ρ= 1.032g/ml
Molecular weight of Tributyrin, MW= 302.36mg/mol
Initial Volume of Substrate, Vs= 1.0ml
Initial Total Volume, VT= 300ml
[ ]
Steps are repeated for Vs= 2.0ml and 3.0ml
REACTION RATE
SUBSTRATE A at 0.5ml enzyme:
(
) (
) (
)
(
) (
) (
)
Similar calculation is carried out for the other enzyme volumes of substrate A and
similarly for substrates B and C. All the calculated values are shown in table 10 .
DETERMINATION OF Km and Vmax values:
LINEWEAVER-BURK MODEL:
For figure 5 (enzyme volume 0.5ml) :
Slope =-1.6478
Intercept =455347
Equation:
[ ]
Similar calculation is carried out for the other enzyme volumes of substrate A and
similarly for substrates B and C.
EADIE HOFSTEE MODEL:
For figure 6 (enzyme volume 0.5 ml):
Slope = 0.00002
Intercept = -2E-07
Equation:
[ ]
HANES MODEL:
For figure 7 (0.5 ml of enzyme):
Slope = 725598
Intercept =-7.2848
Equation: [ ]
[ ]
Similar calculation is carried out for the other enzyme volumes of substrate A and
similarly for substrates B and C.
ENZYME CONCENTRATION
Volume of enzyme, VE
Initial concentration of enzyme,[Eo]
Total volume of solution (substrate + enzyme). VT
[ ]
Concentration of Enzyme, [E] = 24.390LU/m3
Similar calculation is carried out for enzyme volumes of VE=1.0ml and 1.5ml
DETERMINATION OF Kcat VALUES FROM HANES PLOT
Sample calculation for enzyme volume 0.5ml
[ ]
LAB RAW DATA: