Chloroplast Reduction of Indophenol
See previous protocol for the preparation of the purified chloroplasts. Continue to keep them ice cold until the moment you add them to the prepared tubes. Teams of two.
purified spinach chloroplasts, ice cold
0.1 M PO4 buffer, pH 6.5 (pH 7.0 OK?)
2.5 x 10-4 M 2,6 dichlorophenolindophenol
(36.3 mg DCIP/500 mL, A600 = 3.0)
10 uL and 200 uL displacement pipets with tips
seven cuvettes (or unblemished 13×100 mm test tubes)
100 watt light
37C incubator (can exclude light, for whole class)
Set up apparatus:
a: 37C hot block (for 13×100 mm tubes) nearby and warmed up.
b: 37C incubator warmed up.
c: spectrophotometer on the same desk as the light exposure apparatus.
d: 100 watt bulb with reflector 25 cm from open test tube rack.
Construct a data table in your notebook to accept time course data (See below).
Select and label seven cuvettes (or six very clean, unblemished 13 x 100 mm test tubes to serve as cuvettes) (B for blank + six tubes).
Prepare DCIP reaction mix which should have an A600 of 0.400 to 0.600.
Per team of two:
12 mL 0.1 M PO4, pH 6.5 buffer
12 mL 0.5 M sucrose
8 mL 2.5 x 10-4 M DCIP
Dispense 4 mL of the reaction mix to each of your tubes with repeater pipetter.
5. Add all of the following ingredients except for the chloroplasts:
Table for recording DCIP reduction A600 versus time of light exposure.
Copy this table into your notebook prior to starting the chloroplast reduction experiment. Every blank square gets a reading.
6. Prewarm prepared tubes (lacking chloroplasts) in 37C hot block for 2 min.
7. Add diluted chloroplasts to tubes 3 and 5. Mix and read the A600 of 1, 3, and 5 against a water blank (= T0 for dark tubes). Place immediately in a 37C incubator, keep light excluded.
8. Read A600 of tube 2, place in front of the light
9. Add 10 uL of chloroplasts to tube 4, mix well, read A600, place in front of the light, and start the stopwatch.
10. Add 20 uL of chloroplasts to tube 6, read A600, place in front of the light, when the stopwatch reads 30 seconds.
11. At 30 second intervals, alternatively read the A600 of 4 and of 6 for 10 minutes. At 5 minutes, also read tube 2. Read tube 2 again at 10 min. (Keep tubes clean, read in consistent configuration.)
10. At 15 minutes, read A600 of all tubes, including those which were kept in the dark.
11. Plot the absorbency of each tube versus time.
•Discuss the significance of the differences between the various six curves.
Here is a graph of reduction of DCIP by 20 uL of chloroplast suspension