I. THE HILL REACTION

A. Introduction

R. Hill and his colleagues made an important contribution to photosynthesis research when they discovered in the 1930'2 that the process of oxygen release can be separated from CO2 fixation. Hill found that isolated chloroplast fragments can be induced to evolve oxygen if a compound capable of being reduced is provided to a chloroplast suspension in light. In the intact leaf, ferredoxin accepts the electron excited in the chloroplast isolation process used by Hill (and this lab) causes these electron acceptors to be lost; therefore, an artificial acceptor is supplied (i.e. 2.6-dichlorophenol indophenol or DCPIP). DCPIP is blue when oxidized (quinone form), but becomes colorless when reduced to its phenolic state:

The rate of oxygen release in isolated chloroplasts may be determined colorimetrically by measuring the rate of loss of blue color as DCPIP is reduced.

B. Procedure

1. Chloroplast isolation.

a.) Weigh about 8g of spinach leaves that have had midveins and petioles removed. (In order to preserve chloroplast activity these leaves have been kept in the dark and cold since they were purchased.)

b.) Rinse the leaves in ice water, then cut them into pieces about 1cm square. Place the leaf pieces into a pre-chilled blender with 40 ml ice-cold 0.5M sucrose. Blend for 15 sec at top speed, wait about 10 sec, then blend again for 10 sec. Squeeze the leaf slurry through four layers of cheesecloth into a pre-chilled beaker. Pour equal amounts of the filtered, green slurry into two centrifuge tubes (30 ml size). Store the filled tubes in a beaker filled with ice until they can be centrifuged.

c.) Centrifuge the slurry at 200x gravity for 3 min. Unwanted whole cells and cell wall debris are spun to the bottom of the tube at this speed. Decant the supernatant into fresh, cold centrifuge tubes and centrifuge at 1,000x gravity for 7 min. This will spin the chloroplasts down to the bottom of the tube.

Discard the supernatant this time, and resuspend the bottom pellet of chloroplasts into 10 ml of cold 0.5M sucrose, by using a glass rod. After the chloroplasts have been resuspended, combine test tubes and spin again at 1,000x gravity for 7 min to obtain a pellet of moderately pure chloroplasts. Discard the supernatant, and resuspend the pelletin 25 ml of cold 0.1M phosphate buffer at pH 6.5. Keep the chloroplast suspension in an ice bath.

2. Effect of Light Intensity on Chloroplast Activity.

a.) Obtain 6 test tubes and add the reagents as listed in Table 1. Thoroughly mix the contents of each test tube.

b.) Place the colorimeter wave length setting at 600 nm, adjust the infinite absorbance setting without using a colorimeter tube (this will block the colorimeter light path with a shutter), then adjust zero absorbance using a water-filled colorimeter tube. Measure the absorbance of the six solutions after transferring a sample into a colorimeter tube. Return the solution to its original test tube after measuring absorbance. Note: (See instructions on the use of the colorimeter.)

c.) Place the test tubes in a water-filled beaker (to prevent heating from the incandescent lamp), and at the light source distances specified. Place the DCPIP control and the tube wrapped in aluminum foil in water-filled beakers 60 cm from the light source.

d.) Calibrate chloroplast activity by measurement of the absorbance of treatments #3 to #6 after five minutes. If the dye in treatment #3 (closest to the light) has turned colorless, discard all the tubes, and set them up again using 0.5 ml chloroplast suspension. If the absorbance of treatment #3 has not diminished by at least 20%, discard all tubes and remake them, using 2.0 ml chloroplast suspension. Otherwise, continue the light treatment.

e.) Measure the absorbance of treatments #3 through #6 at five minute intervals until most of the dye in treatment #3 is decolorized. Measure the absorbance of all treatments at the end of the experiment, and record the values. Calculate and graph the absorbance for each treatment as a function of time on the first graph of your report. Compute the portion of light reaching the three farthest distances by use of the relationship that light diminishes as the square of the distance. Use 1.0 as the relative intensity at the 30 cm distance. On a second graph, plot the change in absorbance during the first ten minutes as a function of relative light intensity.