RP8 Dehydrogenase activity in chloroplasts

Describe the role of the enzyme dehydrogenase in photosynthesis

● Catalyses the reduction of NADP in the light-dependent reaction

○ NADP accepts (gains) electrons from photoionisation of chlorophyll / photolysis of water

Describe how rate of dehydrogenase activity in extracts of chloroplasts can

be measured

1. Isolate chloroplasts from fresh green leaves (e.g., spinach) using ice-cold buffer and a centrifuge.

2. Set up test tubes as follows:

Tube	Contents	Light Condition
A	DCPIP + isolation buffer (no chloroplasts)	Light
B	DCPIP + chloroplast suspension	Light
C	DCPIP + chloroplast suspension	Dark (wrapped in foil or kept in a dark cupboard)

3. Shine light on test tubes and time how long to it takes for DCPIP to turn from blue (oxidised) to colourless (reduced)

• record how long it takes for solution to turn colourless

4. Rate of dehydrogenase activity (s-1) = 1 / time taken

what is DCPIP? methylene blue can be used too

redox indicator used to measure rate of dehydrogenase activity in LDR

it turns colourless when reduced (gains H and electrons)

faster the colour change, the faster the rate of dehydrogenase activity

Expected Results and Explanation

Tube	Expected Observation	Explanation
A (no chloroplasts)	No colour change – DCPIP remains blue	No chloroplasts = no electron donors = no reduction of DCPIP
B (light + chloroplasts)	DCPIP decolourises rapidly (blue → colourless)	Light excites electrons in chlorophyll → electrons reduce DCPIP
C (dark + chloroplasts)	Little or no colour change	No light = no photoionisation of chlorophyll = no electron transfer to DCPIP

Extending the experiment

The effect of ammonium hydroxide on the time taken for chloroplasts to decolourise DCPIP.

(NH₄OH)?

• increases the pH of the solution.

• It can disrupt thylakoid membranes.

• In high concentrations, it may denature proteins, including enzymes.

• It acts in the same way as many weed killers.

You could add an extra test tube to investigate the effect of ammonium hydroxide on electron transfer:

Chloroplasts + DCPIP + Ammonium hydroxide (DCPIP decolourises more slowly or not at all)

Why This Happens

High pH or membrane disruption may affect the structure of photosystems or electron carriers, reducing the flow of electrons from chlorophyll.

fewer electrons are passed down the ETC, less DCPIP is reduced, so the colour change slows or stops.

Here tube A and tube C both acted as controls. 

Tube A shows that the colour change of DCPIP in tube B is due to the presence of chloroplasts/ chlorophyll.

Tube C shows that light energy is required for the transfer of electrons and the reduction of DCPIP.

by comparing: we can then conclude that DCPIP changed from blue to colourless due to its reduction by electrons and that these electrons came from chlorophyll during the light dependent reaction.

Explain how chemicals which inhibit the decolourisation of DCPIP could slow the growth of weeds.

if chemicals are inhibiting the decolourisation of DCPIP, this indicates they must be inhibiting the transfer of electrons:

1. proton gradient will not be formed. So, protons will not diffuse through the channel in the ATP synthase enzyme and ATP will not be produced

2. electrons will not be transferred to NADP, hence why we do not form NADPH

3. Less / no GP reduced to triose phosphate

Suggest a limitation with the method and how the experiment could be modified to overcome

this.

● End point (colour change) is subjective

● Use a colorimeter

● Measure light absorbance of sample at set time intervals

● Zero colorimeter using the colour standard

Give examples of variables that

could be controlled.

● Source of chloroplasts

● Volume of chloroplast suspension

● Volume / concentration of DCPIP