1. Investigating photosynthesis: using extracts of chloroplasts

2. Using isolated chloroplasts
It is fairly easy to show that plants produce oxygen and starch in photosynthesis.
At KS4 you may have collected the gas given off by Canadian pondweed (Elodea) and tested leaves for starch.
It is not quite so easy to demonstrate the other reactions in photosynthesis.
In this experiment, DCPIP (2,6-dichlorophenolindophenol), a blue dye, acts as an electron acceptor and becomes colourless when reduced.
DCPIP solution is added to isolated chloroplasts allowing any reducing agent produced by the chloroplasts to be detected.
In the cell, NADP is the electron acceptor that is reduced (reduced NADP) in the light dependent reactions and provides electrons and hydrogen for the light-independent reactions.
This reaction was first demonstrated in 1938 by Robert (known as Robin) Hill and is often called the Hill Reaction.

3. How happy are you with the topic?
Photosynthesis
How happy are you with the topic?
I was planning a review of light dependent reaction if need to support the conclusion of this practical
After going through practical!
What do you remember?

4. Light dependent reaction...overview remember!!
Sunlight
2 electrons
Water
Excited chlorophyll
Chlorophyll
Thylakoid membranes chloroplast
Energy released
Photolysis of water
2H H+ + 4e- + O2
ADP ATP
Photophosphorylation
Respiration/
atmosphere
NADP Reduced NADP

5. Why use DCPIP?
In photosystem I in light dependent stage, NADP acts as an electron acceptor (it accepts electrons and a proton released from split water molecules) causing it to become reduced and oxygen is released. This is the Hill reaction (Robert Hill)
The Hill Reaction depends on electrons released during the light- dependent stage of photosynthesis being picked up by the blue electron acceptor DCPIP.
The reaction can only occur if the thylakoid membranes are illuminated as the light-dependent stage stops in the dark.
DCPIP is blue when oxidised (at pH 7.0) and colourless when reduced, so it is possible to monitor the loss of blue colour as an indication that DCPIP has accepted electrons. It can be used to participate in, and monitor, redox reactions.
In this experiment the DCPIP takes the place of NADP, allowing photolysis to continue even when the supply of NADP has been exhausted because the DCPIP can continue to accept the electrons from the electron transport chain
2DCPIP + 2H2O → 2DCPIPH2 + O2

6. Aim: To investigate photosynthesis using isolated chloroplasts (the Hill reaction).
Independent Variable: Light intensity (light/dark) reaching the chloroplast samples. How will we change this?
Dependent Variable: Amount of DCPIP reduced (can be judged based on the colour of the mixture)/ absorbance on colorimeter. Measure every 2 minutes for 10 minutes. Red filter.
Control Variables: How will you keep constant?
Use chloroplasts from the same species of plant – chopped spinach leaves can be used
Amount of chloroplast/buffer solution added to each tube – 5cm³ to each tube
Amount of DCPIP added to each tube – 10cm³ will be added
Amount of time – both tubes will be left for the same amount of time before comparing the colour of the mixture
Same pH used – buffer solution will be added to maintain pH of 7.0 necessary for DCPIP.
The controls:
DCPIP and isolation solution
DCPIP and chloroplast solution wrapped in foil to remove light

7. Risk assessment DCPIP solution can stain, so avoid skin contact.
Wear eye protection.
Ensure the centrifuge has stopped before inserting or removing the tubes.

8. Watch: Hill reaction
om/watch?v=i5eQ8AN_ zKA&t=443s

9. Method
NB Keep solutions and apparatus constantly cold during the extraction procedure, steps 1–8, to preserve enzyme activity. The extraction should also be carried out as quickly as possible.
1 Cut three small green spinach, lettuce or cabbage leaves into small pieces with scissors, but discard the tough midribs and leaf stalks. Place in a cold mortar or blender containing 20 cm3 of cold isolation medium (Sucrose and KCL in phosphate buffer: suitable ionic and osmotic strength) (scale up quantities for blender if necessary). Why is this important?
2 Grind vigorously and rapidly (or blend for about 10 s).
3 Place four layers of muslin or nylon in a funnel and wet with cold isolation medium.
4 Filter the mixture through the funnel into the beaker and pour the filtrate into pre- cooled centrifuge tubes supported in an ice-water-salt bath. Gather the edges of the muslin, wring thoroughly into the beaker and add filtrate to the tubes.
5 Check that each centrifuge tube contains the same volume of filtrate.
6 Centrifuge the tubes for sufficient time to get a small pellet of chloroplasts (10 minutes at high speed should be sufficient).
7 Pour off the liquid (supernatant) into a boiling tube being careful not to lose the pellet. Resuspend the pellet with about 2 cm3 of isolation medium, using a glass rod. Squirting in and out of a Pasteur pipette five or six times gives a uniform suspension.
8 Store this leaf extract in an ice-water-salt bath and use as soon as possible.

10. We want the chloroplasts to be in an isotonic solution: so no real movement of water in/out

11. Why is it all kept cold?
Using ice cold phosphate buffer solution and ice cold sucrose solution is necessary to slow enzyme action and prevent damage to the chloroplasts before the experiment begins

12. Isolating chloroplasts
The process of isolating the chloroplasts will inevitably cause some damage to the chloroplasts
You end up with a mixture of intact chloroplasts and thylakoid membranes without the surrounding stroma and outer membranes.
This is actually an advantage because it means that the DCPIP can access the thylakoid membranes directly, without having to pass through the outer membranes, to accept electrons directly from the electron transport chain.

14. Using the chloroplasts
Read all the instructions before you start.
Note: The DCPIP solution should be used at room temperature.
Set up five labelled tubes as shown: Why do we set these up?
When the DCPIP is added to the extract, shake the tube and note the time.
Place tubes 1, 2 and 4 about 12–15 cm from a bright light (100 W).
(Light intensity could be varied by moving the lamp used for illumination various distances away from the tubes of chloroplasts and DCPIP. The distance of the lamp from the reacting tube could be measured and the actual value of the light intensity can then be calculated using the calculation of 1/ distance squared)
Place tube 3 in darkness.
Note any changes observed in the five tubes. Make any appropriate measurements to allow comparisons between the tubes.
Colorimeter

15. Why have we set these up? Tube 1 (leaf extract + DCPIP)
Electrons produced by chloroplasts in light dependent reaction reduced DCPIP, blue-colourless:
The reaction
Tube 2 (isolation medium + DCPIP)
no colour change (blue) which shows DCPIP does not decolourise when exposed to light.
Control, DCPIP does not change colour without chloroplasts
Tube 3 (leaf extract + DCPIP in the dark/tin foil)
no colour change, loss of colour in tube 1 is due to light on extract
Control, needs light for DCPIP to work as an electron acceptor
Tube 4 (leaf extract + distilled water)
No colour change, extract does not change colour in light
Control: DCPIP needed for colour change
Tube 5 (supernatant + DCPIP)
No colour change if supernatant is clear, some decolouring if slightly green
Control: proving you need the chloroplast extracts in the pellet

16. Operating instructions for a typical colorimeter
Switch on the instrument at least 5 minutes before use to allow it to stabilise.
Select the most appropriate filter for the analysis and insert it in the light path
Colour choice of filter: Why red on a blue/green solution: The blue solution appears blue as all other colours have been preferentially absorbed. The red is the most strongly absorbed
Place the reagent blank solution/or water in the cuvette and zero the instrument (consult your manufacturers instruction about how to do this.)
Place the sample in the colorimeter and read the absorbance of the solution. If the absorbance is "over range" (usually > 2.0) then the sample must be diluted to yield a value within the limits of the instrument.
At intervals, recheck the reagent blank to ensure that there is no drift in the zero value

17. Results & Calculations
Colorimeter with a red filter
As Hill reaction takes place the absorbance will decrease as DCPIP gets reduced and loses its blue colour
The faster the absorbance decreases the faster the Hill reaction
You can plot a graph of absorbance against time (min)

18. Sample results See the handout Using a bench centrifuge
Using a micro centrifuge

19. Conclusion: DCPIP takes the place of NADP in this set-up.
NADP is usually reduced in the light-dependent reaction as electrons are excited from the photosystems and passed along an electron transport chain.
Because this reduction of DCPIP requires light, the sample under a bench lamp should go completely colourless as all the DCPIP is reduced from blue to colourless.
However, the sample in the dark cupboard has exposure to little , if any, light. This results in a lack of reduction of DCPIP and so the solution stays blue overall

20. Evaluation Points:
Test tube A solution does not decolourise (systematic error) – increase light intensity of bench lamp.
Damaged chloroplasts skew results (random error) – carry out repeats and centrifuging plant material at lower speeds.
Difficult to compare colours of both test tubes (random error) – fill a two cuvettes with solutions from each test tube and use a colorimeter to compare percentage absorbance/transmittance.
Experiment is difficult to do in class as has to be kept cold

21. Extension: Application
(a) Explain the practical procedure used could be modified to investigate the effect of light wavelength on the Hill reaction.
(b) Draw a sketch graph to predict the results you would expect in red, green and blue light.
Chlorophyll does not absorb green light.

22. Application:
Set up tubes identical to tube 1 so that chloroplasts are available.
Set up several tubes so that variability of the data can be measured, and a mean and standard deviation calculated.
To investigate the effect of different wavelengths of light place coloured filters between the light source and the reaction mixture. The tubes should be exposed to light of different wavelengths but for the same time, because time affects number of electrons released.
Light intensity should be kept constant because intensity affects number of electrons released.
A colorimeter with a red filter to measure absorbance.
A simple changed colour or not will not be suitable here as all the tubes may change colour, but to a different extent. Colour changes can be quantified using a colorimeter. A filter is used to maximise the proportion of the light absorbed by the solution. In this case the colour intensity to be measured is of blue so a red filter is used.
The theory of complementary colours does not need to be understood by A level biologists but it is important to understand this consequence.

23. A graph of absorbance against time, which should look something like this:

24. Light dependent reaction...overview remember!!
Sunlight
2 electrons
Water
Excited chlorophyll
Chlorophyll
Thylakoid membranes chloroplast
Energy released
Photolysis of water
2H H+ + 4e- + O2
ADP ATP
Photophosphorylation
Respiration/
atmosphere
NADP Reduced NADP

25. The light dependent reaction
On the thylakoids (granum) membranes– involves photosynthetic pigments and electron carriers – located in thylakoid membranes
When a photon of light (particle) is absorbed by chlorophyll – it causes a change in a pair (2 e-) of electrons – they gain more energy (excited state)
This excited state electron however is more unstable
This causes it to be ‘passed’ around
Light energy
2 e-
Chlorophyll
Chlorophyll +
(oxidised)
(electron)
(reduced)

26. Light dependent reaction:
Electrons are captured by electron acceptors
The electrons pass along the electron transport chain in the thylakoid membranes.
The electrons pass from one carrier protein to the next in a series of oxidation (loss)and reduction (gain) reactions, losing energy in the process
Within the thylakoid space, an enzyme catalyses the splitting of water (photolysis) to give oxygen gas, hydrogen ions and electrons.
This is called photolysis
2H2O 4H+ + 4e- +0²

27. Some of the oxygen produced is used by the plant for respiration, the rest diffuses out of the stomata in the leaves into the air
These electrons from water replace those that were emitted from the chlorophyll molecule, so it is no longer positively charged.(reduced)
Energy released as electrons pass along the chain of electron carriers pumps protons (H+) into the thylakoid space where they accumulate
A proton gradient is formed across the thylakoid membranes and they flow through channels associated with ATP synthase enzymes

28. This flow of protons is called chemiosmosis
The protons from the splitting of water combine with the coenzyme NADP to form reduced NADP
The energy made available as electrons pass from one carrier to the next in the electron transport chain is used to synthesise ATP from ADP and Pi
Kinetic energy from the chemical flow is converted to chemical energy in ATP molecules
The ATP is used in the light independent stage.
Making ATP using light energy is called photophosphorylation

29. Cyclic photophosphorylation
Light (700 nm)
PS I
(chlorophyll a)
P 700
Electron is excited to a higher energy level and emitted from the chlorophyll molecule
The electron is captured by an electron acceptor and passed back to chlorophyll a via a chain of electron carries
During this process, enough energy is released to synthesise ATP from ADP and an inorganic phosphate group (Pi) by photophosphorylation
Product – ATP
Uses PS1
P 700 chlorophyll a
Excited electrons are passed to an electron acceptor and back to the chlorophyll molecule from which they were lost: CYCLIC!
No photolysis of water
No reduced NADP
Small amounts of ATP made
This can be used for light independent reaction
Or used by guard cells to bring in potassium ions against concentration gradient, lowering water potential and causing water to follow by osmosis
Causes guard cells to swell and open.

30. Non-cyclic photophosphorylation
Involves PS1 and PS11
So the primary electron acceptor from PSII (which is the first photosystem – it was just discovered second) has the excited pair of electrons
The electrons are passed down an electron transport chain
Energy used to produce ATP (photophosphorylation)
Final acceptor of these electrons is PS1
REMEMBER: PS11 also has the enzyme which causes photolysis of water
Light also strikes PS1: this pair of electrons , along with the H+ in the stroma join with NADP and form reduced NADP
The electrons in PS11 need to be replaced
These electrons come from the photolysis of water
The H+ produced from photolysis of water take part in chemiosmosis to make ATP
Then are taken up by NADP, in the stroma to form reduced NADP.

31. PSII - basically looks a bit like this (not showing PSI)
Light
PSII e- Pc
Electron passed on to PSI Pq
H20
Lumen of thylakoid (has high conc. of H+)
½ O2 + 2H+
Protein complex
Stroma (low conc. H+)
ATP
ADP + Pi

32. PS I (P 700) PS II (P 680) Used to reduce NADP
Reduced NADP is used to reduce GP to TP in Calvin cycle
GP is produced from CO2 fixation by RuBP – catalysed by rubisco
Formation of TP from GP also requires ATP
TP – starting point for CH2O synthesis
PS I (P 700)
Used to reduce NADP
PS II (P 680)
Electrons from PS II are passed onto PS I via electron carriers – generates ATP
Replaces electrons lost by chlorophyll in PS I after excitation by absorption of light
Used in production of NADP
Electrons lost from PS II are replaced by electrons derived from the photolysis of water

33. Plenary: Complete the table
Non cyclic photophosphorylation
Cyclic photophosphorylation
Which photosystem involved
Does reaction involve the photolysis of water?
Which molecule is the electron donor
Which molecule is final electron acceptor
What are the products

34. Plenary: Complete the table
Non cyclic photophosphorylation
Cyclic photophosphorylation
Which photosystem involved
PS1 PS11
PS1
Does reaction involve the photolysis of water?
yes no
Which molecule is the electron donor
water
Chlorophyll a in PS1 P700
Which molecule is final electron acceptor
NADP
What are the products
Reduced NADP, ATP and oxygen
ATP only

35. Plenary