CHLOROPLAST REDUCTION OF INDOPHENOL

©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
 Reagents for reaction mix


This page has been accessed  Counter times since 5 December 2002. 
 18 Nov 1992, 23 Nov 93, 20 Sept 94, 21 Nov 95, 20 Sept 96, 26 Nov 96, 20 Sept 99, 18 Sept 00
From DBF Notebook, C.C. III, p. 84 & CC IX, p. 107
 apparatus set up
<>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.


 
Supplies: Equipment/ team
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 13x100 mm test tubes)
100 watt light

meter stick
37C incubator (can exclude light, for whole class)

hot block, 37C, next to spectrophotometer
spectrophotometer

<> 
Set up apparatus:
a: 37C hot block (for 13x100 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 recordingDCIP 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.
 



tube
mL
Rxn
Mix
volume of 
chloroplasts
added
<>
light
conditions
 Time of exposure in minutes
0 1 2 3 4 5 6 7 8 9 10 15
1
 4.00
 0 dark   x x x x x x x x x x  
2
 4.00
 0 light   x x x x   x x x x    
3
 4.00
 10 uL  dark   x x x x x x x x x x  
4
 4.00
 10 uL light                        
5
 4.00
 20 uL dark   x x x x x x x x x x  
6
 4.00
 20 uL light                        

 

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.

Here is a graph of reduction of DCIP by 20 uL of chloroplast suspension:

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