Bio 241L
Bio 2 Lab - Photoreduction
Kibak
2012
Photoreduction Lab
Photosynthesis Overview – Kibak will review at the beginning of the lab.
Photosystem II P680 primarily passes “photon activated” high energy electrons
down an electron transport chain to make a proton gradient. That proton gradient is
used to make ATP.
Photosystem I P700 primarily passes “photon activated” high energy electrons to NADP to make NADPH.
ATP and NADPH are used to provide the energy and reducing power to convert CO2 to carbohydrate.
A protein called Ferredoxin NADP Reductase (surprising name) gets an electron from ferredoxin and adds
it to NADP as indicated. This causes a proton from solution to bind, adding an “H” to the NADP.
Oxidized
Reduced
Bio 241L
Bio 2 Lab - Photoreduction
Kibak
2012
In this lab we will measure the power of spinach
photosystem I to reduce an NADP analog called
DCPIP (2,6-dichlorophenol-indophenol, MW =
290.08).
As you can imagine from the figure on the left,
we will be measuring the drop in absorbance
(increase in transmittance) of an oxidized DCPIP
solution using a spectrophotometer. Oxidized
DCPIP appears blue (absorbs all reddish-orange
light) and reduced DCPIP is clear.
We will set up four test tubes:
(A) contains phosphate buffer, water, and live chloroplasts (termed a “blank”)
(B) contains phosphate buffer, water, DCPIP solution, “live” spinach chloroplasts (kept in dark)
(C) contains phosphate buffer, water, DCPIP solution, “live” spinach chloroplasts (kept in light)
(D) contains phosphate buffer, water, DCPIP solution, “dead” spinach chloroplasts
□ Prepare a table describing the test tubes and the treatments. Be sure to fill in the volumes.
Phosphate
Buffer
diH2O
DCPIP
Solution
Live
Dead
Chloroplasts Chloroplasts
1 mL
4 mL
-
3 drops
-
light
1 mL
3 mL
1 mL
3 drops
-
-
1 mL
3 mL
1 mL
3 drops
-
light
1 mL
3 mL
1 mL
-
3 drops
light
Lamp
Total
Volume?
(Fill in)
Tube A
Tube B
Tube C
Tube D
CAUTION!
Adding this starts
the experiment!!!
□ Use the attached time course to record % Transmittance at each time point.
□ Look up chemical properties in MERCK manual and look up safety issues using the MSDS (Material
Safety Data Sheet).
Bio 241L
Bio 2 Lab - Photoreduction
Kibak
2012
□ Make sure the spectrophotometer is warming up and set to 605 nm.
□ Set up your “incubation” area including the floodlight, 1 L beaker filled with water to act as heat sink,
test tube rack, and test tubes [SEE BOARD]. Nearby you should also have:
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bottle of water
bottle of phosphate buffer
DCPIP solution
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waste/rinse beaker
gloves
safety glasses
ice bucket, parafilm
□ Observe how to prepare live and dead spinach chloroplasts ☺. Fresh spinach is ground in 0.5 M ice
cold sucrose. Cover the leaves in the blender with a couple of cm of sucrose solution and add about 10 mL
0.1 M phosphate buffer, pH 6.5.
□ Kibak will give you two batches of chloroplasts to keep on ice. You will boil one batch of chloroplasts
for five minutes.
□ At the top rim of the test tubes place small labels A, B, C, & D. Make sure the labels are small and high
enough not to block the light beam in the spectrophotometer. Handle the test tubes only with gloves.
□ Make a sleeve and cap of aluminum foil to keep B in the dark except briefly when in the
spectrophotometer. Light should not be permitted into this test tube as it is one of the controls.
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Prepare four parafilm caps to use for mixing.
To each test tube, add 1 mL of phosphate buffer.
To test tube A, add 4 mL diH2O.
To test tube B, C, & D, add 3 mL diH2O.
To test tube B, C, & D, add 1 mL of DCPIP solution.
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Press the A/T/C button until the spectrophotometer reads % Transmittance (605 nm). Use a plastic disposable
pipette to carefully add 3 drops live chloroplasts (unboiled) to test tube A. Cover the top of test tube A with
parafilm and invert three times to mix. Insert tube A into the sample holder and adjust the instrument to 100%
transmittance by pressing the "zero" button. Test tube A is your blank.

Look at the clock and note the time. When you are ready to take the readings for the experiment, resuspend the
chloroplasts that have settled in your “live” unboiled batch, and add three drops to the sleeved test tube B.
Immediately cover with parafilm and foil, mix (invert three times), remove from foil sleeve, quickly insert into
spectrophotometer’s sample holder, and record the % transmittance as the time 0 reading in your table. Keep track of
time from that moment for that test tube. Rewrap in foil and place in incubation test tube rack. Take and record
additional readings at 3, 6, 9, 12, and 15 minutes.
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Look at the clock and be ready to note the time. Resuspend the unboiled “live” chloroplasts and transfer three
drops to test tube C. Immediately cover with parafilm, mix, record the time and the % transmittance. Return test
tube C to the rack next to test tube B and repeat these steps at 3, 6, 9, 12, and 15 minutes.. These will be the live
chloroplasts that we keep in the light and that should be reducing the DCPIP to make it clear. Therefore we expect
the % transmittance to increase with time.
Bio 241L
Bio 2 Lab - Photoreduction
Kibak
2012

For test tube D use the boiled (dead) chloroplasts and proceed just as with test tube C. Take and record readings
at 3, 6, 9, 12, and 15 minutes.
Waste Procedure
All DCPIP contaminated solutions must disposed of in the container in the hood. Rinse test-tubes with a
small amount of water and dispose of in the container in the hood as well.
DCPIP contaminated tips are to be put in benchtop plastic bag. Glassware should be rinsed, washed well
with lab detergent, rinsed 3x with warm tap water and 1x with diH2O.
Analysis
Please provide the following five short paragraphs, one data table, and one graph, in ONE PAGE FRONT
& BACK for your write-up due one week from today.
 A single paragraph background section that briefly explains photosystem I and how NADP gets
reduced in “real life,” as well as what the chloroplast uses NADPH for.
 An experimental overview that briefly describes how the DCPIP system works as a replacement for
NADP (Why not use NADP?).
 A Methods section that briefly describes WHAT YOU DID (not what happened!). This is not a
copy and paste of the list of tasks in your handout.
 A results section including Data Table containing your readings at the various timepoints and a
graph of the % transmittance for the three treatments over time. This is what happened (as a result
of what you did).
 A short discussion section where you discuss why you think you got the results for the three
treatments that you did.
Materials
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Timers
Boiling Chips
Test Tubes to fit in Spec 20 (6 per group)
Test Tube Holders (2 per group)
Test Tube Racks (1 per group)
Ice buckets (1 per group + instructor)
10 mL serological pipette for buffer (instructor).
1 Liter clear glass beaker (1 per group)
Light source (1 lamp per group)
Spectrophotometer (1 per group)
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Ice
Spinach
0.5M Sucrose ( ~300 mL) on ice
1 M K2HPO4/KH2PO4 Buffer pH 6.5 (>5 mL/group + 30 mL for
instructor)
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250 μM DCPIP (Dichlorophenol Indophenol) 30 mL in light protected
bottle (>3 mL/group)
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Aluminum foil
Bio 241L
Bio 2 Lab - Photoreduction
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Kibak
2012