510 



Leo P. Vernon, Waldo S. Zaugg and Elwood Shaw 



METHODS AND MATERIALS 



Chloroplasts were prepared from either spinach or poke weed (Phytolacca 

 americana ) as previously described ^^', and were stored in a frozen condition 

 for several months with no appreciable loss of activity for the present reac- 

 tion. Chlorophyll a was prepared by chromatography on a powdered sucrose 

 column as described previously ^^^'. PMS was purchased from Sigma Chemi- 

 cal Company, UQg and sodium deoxycholate from Mann Research Labora- 

 tories, and UQ2 was a gift from Merck, Sharp and Dohme Research Labora- 

 tories. Triton X-100 (alkyl phenoxy polyethoxy ethanol) was purchased from 

 Rohm and Haas, Philadelphia. Experiments were performed under anaerobic 

 conditions with Thunberg tubes attached to either one or two absorption cells 

 as previously described ^^K Anaerobic conditions were obtained by alternate 

 evacuation and flushing with Argon gas ^'^' . The reaction mixtures were 

 illuminated through a red filter (Corning No. 2403) by means of a tungsten 

 filament lamp ^^L Absorbancy changes caused by illumination were deter- 

 mined with either a modified Bausch and Lomb 505 spectrophotometer ^^' or 

 a Beckman DB recording spectrophotometer ^^^'. 



RESULTS 



The ability of chloroplasts to catalyze a coupled photooxidation of PMSH2 

 and photoreduction of added UQ2 is shown in Figure 1. For these experi- 



1 2 3 4 5 6 



MINUTES 



Fig. 1. Photooxidation of PMSH2 by 

 poke weed chloroplasts in the pres- 

 ence of UQ2. Prior to illumination 

 PMS was added in the oxidized form 

 and UQ2 was added in the reduced 

 form (reduced by sodium borohy- 

 dride). The reaction mixture was 

 allowed to stand for 5 minutes 

 during which time the PMS was re- 

 duced chemically by UQ2H2. 

 Anaerobic conditions were employed. 

 The reaction system contained in a 

 final volume of 2. ml the following 

 in fimoles: PMS, 0. 1; UQjHj, 0. 2; 

 phosphate buffer pH 6. 6, 100; and 

 DCMU (where indicated), 0. 04. 

 The chlorophyll content of the 

 chloroplasts was 6 pgrams, and the 

 light intensity was 358 milliwatts. 



ments PMS was added initially in the oxidized form and the quinone in the 

 reduced form, resulting in chemical reduction of PMS by the added reduced 

 quinone before illumination was initiated. Upon illumination a rapid increase 

 in absorbancy at 388 mp was observed, indicating oxidation of the added 

 PMSH2. Control experiments in which reduced quinone was omitted and PMS 



