Investigations with phosphatase

Paul Beaumont

Science & Plants for SchoolsHomerton College, Cambridge

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

PhosphatasePhosphatase

Phosphatase enzymes from plants: a versatile resource for post-16 courses

Meatyard, B. (1999), J. Biol. Educ., 33(2), 109-112.

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

The AssayThe Assay

sodiumcarbonate

Phenolphthaleinphosphate

Colourless

phosphatase,

water

Pink

free phosphate+

phenolphthalein

Experimental Experimental OutlineOutline

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 contentsKit contents

Additional materialsAdditional materials

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

ColorimeterColorimeter

R = Reference T = Test

Direction of Beam

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

GerminationGermination

Creating the extractCreating the extract

12 mung beans +

water (15 cm3)Form

suspension

Enzyme extract

Macerate

1 min

Centrifuge

MacerationMaceration

Loading centrifugeLoading centrifugetubetube

CentrifugationCentrifugation

Retain supernatantRetain supernatant

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

Running the assayRunning the assay

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

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

PhosphatasePhosphataseInvestigationsInvestigations

Effect of temperatureEffect of pHPhosphatase levels from different

speciesPhosphatase levels in different locationsEffect of phosphate concentration