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




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
Phenolphthalein
phosphate
phosphatase,
water
free phosphate
+
phenolphthalein
Colourless
sodium
carbonate
Pink
Experimental
Outline

Preparing a standard curve (Part A)

Preparing the enzyme extract (Part B)

Running the assay (Part C)


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 mung beans +
water (15 cm3)
Macerate
1 min
Form
suspension
Centrifuge
Enzyme
extract
Maceration
Loading centrifuge
tube
Centrifugation
Retain supernatant
Running the Assay
10.0 cm3 buffer
(30oC)
Universal Bottle
Enzyme Extract
(2.0 cm3)
Take 1 cm3 and
add to cuvette
Measure
colour
Incubate
(t min)
Remove 1.0 cm3
for reference
Add PPP
(2.0 cm3)
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





Effect of temperature
Effect of pH
Phosphatase levels from different
species
Phosphatase levels in different locations
Effect of phosphate concentration