investigations with phosphatase paul beaumont science & plants for schools homerton college,...
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Investigations with phosphatase
Paul Beaumont
Science & Plants for SchoolsHomerton College, Cambridge
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OCR specificationOCR specification
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.
5.1 Module 2801: Biology Foundation
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PhosphatasePhosphatase
Phosphatase enzymes from plants: a versatile resource for post-16 courses
Meatyard, B. (1999), J. Biol. Educ., 33(2), 109-112.
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PhosphatasesPhosphatases
Found in wide range of plant and animal tissues
Release phosphate groupsPhosphate available for ATP and
nucleotide synthesis, membrane construction.
Two main groups – acid and alkaline
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The AssayThe Assay
sodiumcarbonate
Phenolphthaleinphosphate
Colourless
phosphatase,
water
Pink
free phosphate+
phenolphthalein
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Experimental Experimental OutlineOutline
Preparing a standard curve (Part A)
Preparing the enzyme extract (Part B)
Running the assay (Part C)
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PPP = phenolphthalein phosphate (substrate)
PP = phenolphthalein (product)
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Kit contentsKit contents
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Additional materialsAdditional materials
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Standard curveStandard 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
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ColorimeterColorimeter
R = Reference T = Test
Direction of Beam
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Standard curveStandard curve
Standard Curve
00.20.40.60.8
11.21.4
0.00E+00 2.50E-05 5.00E-05 7.50E-05
Concentration of PP, mol dm-3
Absorbance, 500 nm
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GerminationGermination
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Creating the extractCreating the extract
12 mung beans +
water (15 cm3)Form
suspension
Enzyme extract
Macerate
1 min
Centrifuge
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MacerationMaceration
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Loading centrifugeLoading centrifugetubetube
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CentrifugationCentrifugation
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Retain supernatantRetain supernatant
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Running the AssayRunning the Assay
Measurecolour
Take 1 cm3 and
add to cuvette
Incubate
(t min)
Universal Bottle
10.0 cm3 buffer
(30oC)
Enzyme Extract
(2.0 cm3)
Add PPP
(2.0 cm3)
Remove 1.0 cm3
for reference
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Running the assayRunning the assay
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ResultsResultsEffect of pH on mung bean phosphatase
activity
0
0.05
0.1
0.15
0.2
0.25
2 3 4 5 6 7 8 9
pH
Absorbance
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ResultsResults
Effect of temperature on mung bean phosphatase
0
0.1
0.2
0.3
0.4
0 20 40 60 80
Temperature , oC
Relative activity
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PhosphatasePhosphataseInvestigationsInvestigations
Effect of temperatureEffect of pHPhosphatase levels from different
speciesPhosphatase levels in different locationsEffect of phosphate concentration