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Aim:
Introduction:
Cofactors are helper molecules which bind to the enzyme tightly and they
help in transformation. NADPH is used as cofactor for CYP P450 which
metabolizes Aminopyrine.
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Enzyme kinetics plays a very important role in interpreting the results, some
of the important parameters are Vmax, Km.
Vmax is the maximum velocity of the reaction at which all enzymes get
saturated by substrate. Vmax give information about how fast the enzyme
can convert substrate to product when it is completely saturated with
substrate (Robert et. al., 2003)
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Figure2. Showing the metabolism of Aminopyrine by Cytochrome P450 2B1 resulting in the
formation of formaldehyde as the product. (pubchem compounds,2009)
Objective:
Materials Required:
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Protocol:
Microsome preparation
Buffers were pre chilled to 4 C and the isolation procedure was carried
out on ice. The liver were excised into washing buffer then
homogenized using homogenizing buffer using motor driven pestle and
tube.
The supernatant obtained was discarded and the pellet was re-
suspended in the homogenizing buffer and centrifuged at 180,000g for
further 60 minutes at 4°C. The pellet was re-suspended in storage
buffer and stored at -80°C.
Carbon monoxide was gently bubbled through the sample only for 30
seconds and few grains of sodium dithionite were added to both
sample and reference cuvettes. After a gentle mixing different
spectrum was obtained with an absorbance peak at 450nm.
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1. 8 sample tubes were marked from 1-8 and solutions were added in
following order as shown in the table below.
Sample tube 1 2 3 4 5 6 7 8
number
Vol. of 50 50 50 50 50 50 50 50
microsomes (μl)
2. All tubes were then vortex mixed and placed in shaking water bath at
37 C. Each tube was placed in water bath after an interval of 30
seconds.
3. After the initial pre-incubation of 4 minutes the reaction was initiated
by the addition of Aminopyrine in the amounts mentioned in the table
below.
1 2 3 4 5 6 7 8
4. All tubes were vortex mixed and were placed in water bath again.
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5. At the end of the 30 min incubation period of each tube, the reaction
was terminated by the addition of 0.5ml 20% TCA and sample tubes
were vortex mixed.
6. Once the reaction has been terminated, sample was centrifuged at
4000 rpm for 5 minutes to precipitate the microsomal protein.
Standard No. S1 S2 S3 S4 S5 S6
8. 0.5 ml of 20% TCA was added to all the tubes and vortex mixed.
Nash Reaction
9. 8 set of fresh tubes were labeled for sample and 6 set of fresh tubes
for standard.
10. 1.5ml Nash reagent was added to all the tubes.
11. 1.5ml of sample and standard supernatant was pipette to the
corresponding tubes containing Nash regent. All tubes are vortex
mixed.
12. Standard and sample tubes were incubated for 10 min in hot water
bath at 60 C and then allow it cool at room temperature.
13. Absorbances of the solutions are measured spectrophotometrically at
412nm using distilled water as reference (refer table 1 & graph 1).
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Protocol
1. 8 plastic tubes were marked and BSA and sodium hydroxide was
added in amounts as mentioned in the table below.
0 1.0 0
0.1 0.9 25
0.2 0.8 50
1.0 0 250
2. In a new plastic tube, microsomes were taken and the quantity was
diluted 200 times using 0.3M NaOH. Here 10 μl microsomes were
diluted with 2000 μl or 2.0 ml of 0.3M NaOH.
3. From the above tube 1 ml of the diluted sample was taken into
plastic tubes in duplicate.
4. Then 2 ml reagent A was added to all the tubes and vortex mixed
and left at room temperature for 10 minutes.
5. Then 2 ml of the given reagent B was added to all tubes and then
vortex mixed and left at room temperature for 10 minutes.
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Results
Formaldehyde calculations
Table 1. Showing standard formaldehyde concentration, absorbance of
formaldehyde at 412nm and corrected values to fit data that in out of range
1 0 0.07 0
2 31 0.105 0.035
3 62 0.176 0.106
4 92 0.212 0.142
5 123 0.255 0.185
6 154 0.339 0.269
7 - - -
8 - - -
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Calculation
y= 0.001x – 0.008
y+0.0080.001 = x
0.038 + 0.0080,001 = x
x = 46
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Table 3.showing Final BSA concentration and the absorbance of samples at 690nm
and corrected values to fit data that in out of range
1. 0 0 0
2. 25 0.001 0
3. 50 0.113 0.112
4. 100 0.210 0.209
5. 150 0.331 0.330
6. 200 0.416 0.415
7. 225 0.509 0.508
8. 250 0.620 0.619
Sample 1 (control) - 0.011 -
Sample 2 (control) - 0.12 -
Sample 1 (PB- - 0.015 -
induced)
Sample 2 (PB- - 0.017 -
induced)
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– In control samples
y = 0.002 x - 0.025
x = y+0.0250.002
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This value is then multiplied with dilution factor which was 200
=3650 μg/ml
=3.65 mg/ml
= 3.65 mg x 50 μl1000 μl
= 0.1825 mg of Protein
x = y+0.0250.002
This value is then multiplied with dilution factor which was 200
=4100 μg/ml
=4.1 mg/ml
= 4.1 mg x 50 μl1000 μl
= 0.205 mg of Protein
Calculation of Velocity
Velocity of the reaction= concentration of formaldehydeduration of the
reaction X protein content(from Lowry'assay)
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in control Or in PB- Or
induced
samples HCHO concentration HCHO concentration
nmol/ml30minX 0.1825 mg samples nmol/ml30minX 0.205 mg
nmol/ml
nmol/ml
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Vmax -Maximum velocity at which enzyme gets saturated, Vo- Initial velocity, Km- shows
the affinity of the enzyme towards substrate
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Vmax -Maximum velocity at which enzyme gets saturated, Vo- Initial velocity, Km- shows
the affinity of the enzyme towards substrate
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Vmax -Maximum velocity at which enzyme gets saturated, Vo- Initial velocity, Km- shows
the affinity of the enzyme towards substrate
From the Lineweaver Burk Plot (graph 5), we obtain : y = 0.004 x + 0.060
eq. 1
From the Lineweaver Burk Plot we know that, x = -1/Km value of y=0
eq. 2
Km = -0.004-0.06
Km = 0.06 mM
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From the Lineweaver Burk Plot we know that, y = 1Vmax and x=0
Vmax = 16.66
nmol/ml/mg
Apparent Vmax for competitive inhibition is same as Vmax so, Vmax’ = 16.66 nmol/
min /ml
Apparent Km cannot be calculated without the Inhibitor concentration and binding constant [Ki] but
for the competitive inhibition apparent Km is lower than the real Km
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Vmax -Maximum velocity at which enzyme gets saturated, Vo- Initial velocity, Km- shows
the affinity of the enzyme towards substrate
From the Lineweaver Burk Plot (graph 6), we obtain : y = 0.002 x + 0.036
eq. 1
From the Lineweaver Burk Plot we know that, x = -1/Km value of y=0
eq. 2
Km = -0.002-0.036
Km = 0.05 mM
From the Lineweaver Burk Plot we know that, y = 1Vmax and x=0
Vmax = 27.77
nmol/ml/mg
Apparent Vmax for competitive inhibition is same as Vmax so, Vmax’ = 27.77 nmol/
min /ml
Apparent Km cannot be calculated without the Inhibitor concentration and binding constant [Ki] but
for the competitive inhibition apparent Km is lower than the real Km
But we know, protein obtained from Lowry method as 0.1825mg for control
and 0.205mg for PB induced samples.
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In Control
In Phenobarbital-induced samples
Km 0.06mM 0.05mM
Discussion :
From the values of total cytochrome P450 in both control (0.632 nmol/mg)
and Phenobarbital (0.874 nmol/mg) it is clear that the Phenobarbital is an
inducer of the enzyme CYP which could be proved by comparing the amount
of total enzyme obtained in both the samples. We can also see a huge
difference in the Vmax of both the samples denoting the high amount of
enzymatic activity in presence of Phenobarbital than in controlled samples.
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The comparative graph above shows that the Km & Vmax of PB induced
(0.05mM) & (27.77nmol/min/mg) is different from the Km & Vmax
(0.06mM) (16.66 nmol/min/mg) of the control. There is big difference in
Vmax of both the sample denoting Phenobarbital as a potent inducer of Cyp
2B1. We got Km of PB induced as 0.05mM which is not significantly lower
than 0.06mM of control. So, we can conclude that it has approximately equal
affinity for its substrate and will reach Vmax/2 faster than the control sample
and Vmax of PB induced samples are much higher than control samples
(difference of 11.11 nmol/min/mg).
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Ratio of Vmax/ Km gives the efficiency of the enzyme. But efficiency of the
enzyme is limited by the rate of diffusion of substrate towards the enzyme
(Campbell, 1995).
Extrapolation of the graph was done to fit the values that were out of the
range.
Also to note that some error may be present in the values due human error,
error in pipetting, variation in the incubation time and also cofactor mixture
was insufficient. So the volume was made using buffer. All these factors have
contributed to the error in the values.
The study published by Rowland and Tozer, suggested that the enzyme
induction in the metabolism of those drugs which have high hepatic
extraction ratio will have complications when drug is given orally than when
drug is given intravenously (Rowland & Tozer,1995).
References:
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