Transcript Document

Investigations with phosphatase
Paul Beaumont
Science & Plants for Schools
Homerton College, Cambridge
OCR specification
5.1 Module 2801: Biology Foundation
5.1.3 Enzymes – Learning Outcomes:
(c) describe and explain the effects of pH,
temperature, enzyme concentration and substrate
concentration on enzyme action.
(d) follow the time course of an enzyme-catalysed
reaction by measuring rates of formation of
products or rate of disappearance of substrate
(e) explain the effects of reversible inhibitors (both
competitive and non-competitive) on the rate of
enzyme activity.
Phosphatase
Phosphatase enzymes from
plants: a versatile resource for
post-16 courses
Meatyard, B. (1999),
J. Biol. Educ., 33(2), 109-112.
Phosphatases
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Found in wide range of plant and
animal tissues
Release phosphate groups
Phosphate available for ATP and
nucleotide synthesis, membrane
construction.
Two main groups – acid and alkaline
The Assay
P h e n o lp h th a le in
p h o s p h a te
p h o s p h a ta s e ,
w a te r
fre e p h o s p h a te
+
p h e n o lp h th a le in
C o lo u rle s s
so d iu m
ca rb o n a te
P in k
Experimental
Outline
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Preparing a standard curve (Part A)
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Preparing the enzyme extract (Part B)
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Running the assay (Part C)
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PPP = phenolphthalein phosphate
(substrate)
PP = phenolphthalein (product)
Kit contents
Additional materials
Standard curve
Solution
Number
Volume of
PP stock,
(cm3)
Volume
water,
(cm3)
Volume
Na2CO3
(cm3)
PP
concentration
(mol dm-3)
1
0
5
5.0
0.0
2
1.0
4.0
5.0
5.0 x 10-6
3
2.0
3.0
5.0
1.0 x 10-5
4
3.0
2.0
5.0
1.5 x 10-5
5
4.0
1.0
5.0
2.0 x 10-5
6
5.0
0.0
5.0
2.5 x 10-5
Colorimeter
Direction
of Beam
R = Reference
T = Test
Standard curve
Absorbance, 500 nm
Standard Curve
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0.00E+00
2.50E-05
5.00E-05
7.50E-05
Concentration of PP, mol dm -3
Germination
Creating the extract
12 m ung beans +
M a c e ra te
3
w a te r (1 5 c m )
1 m in
F o rm
s u s p e n s io n
C e n trifu g e
E nzym e
e x tra c t
Maceration
Loading centrifuge
tube
Centrifugation
Retain supernatant
Running the Assay
1 0 .0 c m
3
b u ffe r
o
(3 0 C )
R e m o v e 1 .0 cm
U n iv e rsa l B o ttle
E n z y m e E x tra ct
fo r re fe re n c e
3
(2 .0 c m )
T a k e 1 cm
3
and
a d d to c u v e tte
M e a s u re
c o lo u r
In c u b a te
(t m in )
Add PPP
3
(2 .0 cm )
3
Running the assay
Results
Effect of pH on mung bean phosphatase
activity
Absorbance
0.25
0.2
0.15
0.1
0.05
0
2
3
4
5
6
pH
7
8
9
Results
Effect of temperature on mung bean
phosphatase
Relative activity
0.4
0.3
0.2
0.1
0
0
20
40
Temperature , o C
60
80
Phosphatase
Investigations
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Effect of temperature
Effect of pH
Phosphatase levels from different
species
Phosphatase levels in different locations
Effect of phosphate concentration