486 W. D. Bonner, jr. 



3. Reduction with DPNH in the presence of n-heptyl-8-hydroxy-quinoUne- 

 N-oxide (HOQNO). 



4. Reduction with sodium dithionite. 



It will be seen below that these four methods of treatment coupled to the 

 low temperature absorption spectrophotometry represents a powerful 

 approach to the study of plant cytochromes. 



Homogenate 

 2000 g X 10 min 



Supernafant 

 Discard 10,000 g x 20 min 



Resuspend in 0-5 M iO.OOO g X 20 min 



mannitol/phospiiate 

 and tesediment / 



"mitochondria" ... . /\ ^ ... 



Discard 30,000 g x I hour 



Sediment / \ Supernatant 



"Microsomes Discard 



Fig. 2. Preparation and fractionation of plant tissue homogenates. 200 g of 

 plant tissue was ground in 200 ml of 0-5 m mannitol, 001 m phosphate (pH 7-3) 

 and 0-1% of cysteine or glutathione and 10"^ m versene. The material was 

 either hand-ground in mortar and pestle with sand, or disintegrated in a Waring 

 blender run at a low speed. The ground material was squeezed through muslin, 

 giving the homogenate. 



In the following, 'component 558' is used to indicate the cytochrome having 

 an a-absorption band at — 190°C in phosphate buffer at 558 m//, and other 

 cytochromes are indicated by a corresponding notation. 



As shown by Okunuki (1939), pollen is a material relatively rich in cyto- 

 chromes. Dithionite-reduced pollens derived from a variety of sources show 

 three well defined absorption bands when studied with a microspectroscope. 

 The low temperature difterence spectrum, resulting from the difference in 

 absorption between a well aerated corn pollen suspension and a dithionite- 

 reduced suspension is shown in Fig. 3. It can be seen that this spectrum shows, 



