LABORATORY 6

6.1

Pre-lab Assessment Sheet for LABORATORY 6

Family Name: First name: Bench No: Stream No: Mark: Date:

Questions/Problems for presentation to group:

You are required as your preparation for lab 6 to construct a powerpoint presentation about the problem in the box below: Photosynthesis, fermentation, fizzy drinks and rubbers. What is the common link? All free energy consumed by biological systems arises from solar energy that is trapped by the process of photosynthesis. The basic equation of photosynthesis is simple: H2O + CO2 (CH2O) + O2 but the mechanism of photosynthesis is relatively complicated. In 1772 the first experiments which led to the identification of "carbon dioxide" and "oxygen" as gases involved in respiration were performed. Who was the researcher who performed these experiments? Describe the experiments leading to the identification of these "gases"? How were these results extended by Jan Ingenhousz in the late 1700's? What do photosynthesis, fermentation, fizzy drinks and rubbers have in common?

Your power point presentation must be: 6 slides maximum No more than 4 bulletpoints per page All text must be no less than 28 point font Images are not necessary but welcome You must bring your six slides printed onto 1 page for your pre-lab to be marked. As part of your lab assessment be pre pared to discuss your powerpoint as a group of 4 students Presentation (4 marks) Note: The purpose of this assessment is to test your ability to research, summarise, and present a large amount of information concisely. This is a skill you will need to develop no matter what vocation you may choose.
LABORATORY GUIDE BIOSCI 101

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LABORATORY 6

BIOSCI 101

LABORATORY GUIDE

LABORATORY 6

6.3

LABORATORY 6 Energy Capture and Storage

It is important to become familiar with the main biochemical events of photosynthesis and to be able to localise these events within the detailed structure of the appropriate organelle. In this practical, you will study the light-capturing reactions of photosynthesis by measuring the reduction of a dye which acts as an electron receptor.

ASSESSMENT
1. Presentation of your powerpoint to group: The tutor or a demonstrator will be present for your presentation. 2. Graphs: Initial velocity of electron flow and Relationship between electron flow and light intensity. Suggested reading in Campbell Biology: 9th & 8th edn. Chapter 10.

Photosynthesis and electron transfer In chloroplasts sunlight supplies energy to initiate an electron flow. ATP is made as these electrons are passed along a transport chain, and the coenzyme hydrogen/electron carrier, NADP+, is reduced. The ATP and the NADPH produced in the light-capturing reactions go on to supply energy and electrons to drive the Calvin Cycle in which carbon dioxide is fixed into carbohydrate. Meantime, the electrons lost from the chlorophyll are replaced by those obtained from the photolysis of water. Chloroplast membranes provide a fabric which contains organised sequences of electron carriers. Membranes of both the chloroplast and mitochondrion also form closed compartments for the accumulation of protons, thus allowing the creation of proton gradients essential to ATP production by chemiosmosis. In 1937, Robin Hill found that Photosystem 1 chloroplasts extracted from leaves are able to produce oxygen when 2eilluminated and that oxygen production NADP+ can be increased by providing an artificial Photosystem 11 2eNADPH electron acceptor. This system illustrates 2ea kind of incomplete photosynthesis DCPIP involving the light-capturing reactions P700 2e only, without the CO2 fixation. In the DCPIPH2 chloroplast the natural supply of NADP+ able to be reduced to NADPH is small, Sun H2O as the molecules shuttle back and forth Electrons diverted to P680 a carrier sustitute 1/2 O2 + 2H+ between the electron transport chain and the Calvin Cycle. We will supply an excess of substitute hydrogen/electron Sun The Light-Trapping Reactions sink to prolong the electron flow for a (Source: Vivian Ward, SBS) relatively long period of time.
LABORATORY GUIDE BIOSCI 101

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LABORATORY 6

We will use for this purpose the redox dye 2,6-dichlorophenol indophenol (DCPIP) which is a blue colour when oxidised and colourless when reduced. This will allow us (using a spectrophotometer) to follow the progress of bleaching during electron flow: Light DCPIP + 2e- + 2H+ > DCPIPH2 (blue) Chloroplasts (colourless) In the experiment, our active photosynthetic material is a suspension of chloroplasts extracted from either spinach or silverbeet leaves (depending on market freshness). Overall Strategy 1. Work in pairs. 2. Each pair will measure the reduction of dye over a standard set of time periods using one particular light intensity. A cuvette containing the reaction mixture is placed at a chosen distance between 10 and 40 cm from the light source. 3. Light exposure intervals of 30 sec (half minute) will be used between absorbance readings. These will continue either for ten readings or until the absorbance reaches a repeated minimum value (whichever happens the sooner).

The Spectrophotometer Before proceeding you should familiarise yourself with the principles of colorimetry and the use of the spectrophotometer. This is described at the beginning of the Course Guide under Laboratory Information.

4. A graph will be made of the disappearance of colour as a decrease in absorbance over the progressively increasing periods of exposure. (Graph 1) 5. Graph 1 will be used to determine the initial velocity of electron flow which corresponds to your particular light intensity. To do this, a straight line of best fit is drawn passing through your first measurements, i.e. a line of initial slope. 6. The drop in absorbance per minute can then be read from this line. Write your answer on Graph 1, showing your calculations. 7. You will then use the class means for initial velocities of electron flow (= absorbance drop per minute) to plot the relationship between electron flow (y axis) and decreasing distance from the light source (= increasing light intensity) (Graph 2).

BIOSCI 101

LABORATORY GUIDE

LABORATORY 6

6.5

The chloroplast suspension has been prepared by homogenizing silver beet leaves in 0.5 molL-1 sucrose buffered at pH 6.5 for 1 min, then filtering through cheesecloth. The concentration of chloroplasts is then adjusted with the buffered sucrose. The green color of the silver beet chloroplast suspension is registered as absorbance by the spectrophotometer. We therefore use a reference blank with chloroplasts without dye to set the spectrophotometer to read zero absorbance by pressing CAL. Then when we measure the reaction tube, we know that the absorbance registered is that of the dye alone. We have also chosen the wavelength to correspond with an absorbance peak for the blue dye but not for the chlorophyll. 1. Keep chloroplasts on ice and in the dark until needed. 2. Make a note of what the distance at which you will conduct the experiment is, this is marked on the tape on your bench. 3. Make sure you are familiar with how to start, stop, and reset the timer 4. You will need five cuvettes, and two test tubes. Please do not write on the cuvettes 5. Fill in the suggested volumes in the method table (see blackboard). Check use of micropipettor. Table of mixtures for the photosynthesis experiment Tube Chloroplasts Buffer Distilled H2O A Reference blank B Reaction mixture light C Reaction mixture dark control D Dye only 3 ml 3 ml 3 ml
not required
(Check with tutor) not required

DCPIP Total volume 0 _____ ml _____ ml _____ ml _____ ml

not required

(Check with tutor) not required

not required

(Check with tutor) not required (Check with tutor) not required

3 ml

6. Prepare a water bath by filling the 250 ml beaker around 2/3rds full with room temperature water from the tap. Cover this with foil so that it is dark inside 7. The cuvette holders have places marked for A, B, C, and D. Use these holes for the corresponding samples. NEVER mark the cuvettes 8. Ensure spectrophotometer is available for you to use uninterrupted for 20 mins. Ensure that the wavelength is set to 620 nm. 9. At all times ensure your cuvettes are free from condensate as this will interfere with the readings. Procedure for Cuvettes A and B. 10. Prepare two cuvettes A) with the suggested vol of dist water B) with the suggested vol of blue DCPIP dye

11. Put 7 mls cold chloroplast suspension into a glass test tube, place this inside your dark water bath and set the timer for 5 mins. 12. In the mean time zero the spectrophotometer with a cuvette of 3 mls dist
LABORATORY GUIDE BIOSCI 101

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LABORATORY 6

water. The next few steps must be done in quick succession. 13. Cuvette A: When the 5 mins is up, take 3 mls warmed chloroplast and add to the cuvette with water. The amount of water added is the same as the amount of DCPIP (but check this with your tutor). Quickly mix gently by inverting cuvette once with parafilm held over the top with your finger. Place into the spectrophotometer, wait till the Abs is stable (about 1-5 secs) and press CAL. This zeroes the spectrophotometer. Take cuvette out of the spectrophotometer. 14. Cuvette B: Now take 3 mls of the warmed chloroplasts and add to the cuvette with the blue dye. Invert gently once using the parafilm and put into the spectrophotometer. Read the immediate Abs and record in the table provided. 15. Quickly place this cuvette in front of the light source at the correct distance from the light bulb, with the clear side of the cuvette facing the light. Immediately start the timer 16. When 30 secs has gone by, put this cuvette back into the spectrophotometer and read the immediate Abs and record in the table. Quickly put it back in front of the light, and: stop, clear and start the timer. Once started keep measurement time in the spectrophotometer to a minimum and return the cuvette to its light exposure without delay. 17. Again once 30 secs has gone by, put it into the spectrophotometer, read Abs and replace in front of the light, restart the clock. 18. Do this 10 times so that the cuvette has been in front of the light a total of 5 mins. Minimize delays. Your chloroplasts are deteriorating all the time and also the decolourisation of the DCPIP due to light exposure is reversible, especially in the dark. Procedure for Cuvette C (Dark control) 19. Prepare one cuvette C with the suggested vol of blue DCPIP dye 20. Warm 4 mls of the chloroplasts for 5 mins in your water bath in the dark 21. Take 3 mls of the chloroplasts and add to cuvette C, gently invert, place in the spectrophotometer and record the Abs in the results table. 22. Enclose the cuvette entirely with foil, place in front of the light and start the clock. Leave for 5 mins then record the Abs, after taking the foil cover off Procedure for Cuvette D (Dye only control) 23. Prepare one cuvette D with the suggested vol of blue DCPIP dye 24. Before doing cuvette D, you will need to re zero the spectrophotometer using a fifth cuvette containing 3 mls of buffer. Press CAL. 25. Add another 3 mls of buffer to cuvette D, invert to mix and read the initial Abs. Record in the table 26. Place in front of the light for 5 mins, and then read the Abs. Record in the table. 27. Proceed to drawing graphs and answering questions.

BIOSCI 101

LABORATORY GUIDE

LABORATORY 6

6.7

Membranes and internal components of the chloroplast Add labels to show: 1. Location of Calvin cycle enzymes. 2. Where H+ from light reaction accumulates. 3. Location of electron carriers. 4. Location of photosystem pigments. 5. Location and direction of H+ driven ATP synthetase channels.
(From: Wolf, S. 1981 Biology of the Cell 2nd ed. Wadsworth)

Table of results for Cuvette B Accumulated No. Time in Front Absorbance No. of Light 0 2 8 3 9 4 5 10 6 Accumulated Time in Front of Light Absorbance

1 7

Initial Absorbance Cuvette C (Dark Control) Cuvette D (Dye Only)

Final Absorbance

LABORATORY GUIDE

BIOSCI 101

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LABORATORY 6

Photosynthesis
Chemical Sucrose DCPIP Formula C12H22O11 C12H7NCl2O2

Hazardous Substances Hazard Additional information Inhalation of powder may cause respiratory irritation. 2, 6-Dichloroindophenol. Cause irritation to eyes skin, respiratory system and digestive tract. TOXICOLOGY NOT FULLY INVESTIGATED. Maybe harmful if swallowed.

Irritant

Di-Sodium Hydrogen Phosphate Sodium diHydrogen Phosphate

Na2HPO4 NaH2PO4 Irritant

Eye and skin irritant.

CLEANING UP INSTRUCTIONS
1. Place a) cuvettes b) colour coded test tubes c) chloroplast flasks d) blue/white tips in labelled containers by sink. 2. Place glass test tubes in bin on trolley by sink. 3. EMPTY ice bucket and stack by sink. 4. Stack white tray on side bench.

BIOSCI 101

LABORATORY GUIDE