376 



C.P. i^ittinghara and P.M. Bishop 



shov7s that, as the dark period between the two flashes is in- 

 creased, the yield from the long flash rises slowly to a maximum; 

 this occurs when the two flashes are about 10 sec. apart. The 

 yield remains relatively constant up to 15 sec, separation, and 

 then declines gradually. There is still some influence of the 

 first flash on the second when the two are 25 sec. apart. A very 

 similar plot is obtained when ferricyanide is reduced in the ab- 

 sence of phosphorylating reagents. 



As may be observed from fig. 1, the addition of an uncoupling 

 agent to a ferricyanide system has the effect of shortening the 

 dark time necessary between the two flashes to obtain the maximum 

 yield from the second flash. The yield from the long flash now 

 rises rapidly as the dark time between the short and the long 

 flash is increased; maximum yield is obtained when the two are be- 

 tween 1.5 and 2.5 sec. apart. With further increase of the dark 

 time between the flashes, the yield declines exponentially to 

 about 10 sec. separation time; following this there is a further 

 slight rise with a maximum at 15 sec. and then a further decline, 

 until at 20 sec. there is only a slight effect of the first flash 

 on the second. Figure 1 also shows that very similar results are 

 obtained when catalytic amounts of TPIP are added to ferricyanide 

 and when TPIP alone is used as an oxidant. The maximum increase 

 in the yield of the long flash is obtained when the two flashes 

 are between 0.5 and 3 sec. apart; there is a similar secondary 

 peak occurring when the two flashes are separated by a dark inter- 

 val of about 14 sec. 



The data presented above demonstrate that the time course of 

 the flash effect varies according to the nature of the oxidant 

 system. The results can be separated into two groups according 

 to whether the maximum yield is obtained when the two flashes are 

 separated by a long time interval - as found with ferricyanide in 

 the presence or absence of phosphorylating reagent, or by a short 

 one - as occurs with TPIP and on the addition of TPIP or of an 

 uncoupling agent to the ferricyanide system. The size of the in- 

 crease at its maximum is largest in the latter group. 



Monochromatic flash illumination with monochromatic continuous 

 background light 



The results obtained from a typical series of experiments with 

 chloroplasts reducing ferricyanide in the presence of phosphory- 

 lation cofactors, are presented in figure 2. From the figure it 

 can be seen that there are two regions where background light is 

 most effective in enhancing the yield from the 697 m^i flash; one 

 extending from 642 to 660 mp, has a broad peak from 646 to 653 mjj; 



