72 



Britton chance and Walter D. Bonner, Jr. 



as well as a slow time scale permits the observation of the rela- 

 tively instantaneous fluorescence change as separate from the 

 kinetics of the P70O oxidation. 



Steady state illumination allows the possibility of the time 

 discrimination between the fluorescence and absorbancy changes 

 as the fluorescence change woiold occur in a short time, while 

 absorbancy changes, as clearly indicated in the charts, requires 

 about half a minute to reach the steady state level. We have, 

 therefore, reasonable assurance that the double-beam method 

 guards against the very annoying fluorescence due to chlorophyll 

 in emission, in the region of TOO miji. 



When the double-beam spectrophotometer is used with intense ac- 

 tinic illumination of fluorescent materials, where the average 

 photocurrent may increase greatly during actinic illumination, it 

 is necessary to connect the photomultiplier output directly to the 

 ac amplifier, bypassing the chopper contacts used for calibraUon 

 or for single- ended operation. However, a doubling of the aver- 

 age photocurrent causes no difficulties. 



EXPERD^[SM!AL RESULTS 



A comparison of the light induced kinetics at room and low temp - 

 eratures. 



Figure 3 illustrates room and low temperature kinetics of a 

 Swiss chard leaf upon illumination with 68O m\i actinic light. 

 This comparison is facilitated by the possibility of measuring 

 the room temperatvire kinetics before filling the De>ra,r flask with 

 liquid nitrogen. On the left hand portion are the room tempera- 

 ture kinetics which are seen first to be rather small in amplitu* 

 in comparison with the low temperature kinetics. (The rate of 

 reaction is apparently more rapid at the low temperature in spite 

 of the decrease of sensitivity to absorbancy changes at the low 

 temperatures.) Two effects are undoubtedly involved. First, an 

 enhancement of the absorbancy change due to increased light scat- 

 tering and a sharpening of the cytochrome band occvirs. Second, 

 the dark reduction of cytochrome f which, at room temperature is 

 almost identical in rate to the oxidation, does not occur, and 

 hence, the correct velocity constant for cytochrome f oxidation 

 can be obtained at the lovr temperatui'e. This diagram suggests 

 that measurements of quantum requirements for cyi:ochrome reac- 

 tions may be more acctirately measured at the low temperatures (ll) 



Difference spectrum for illumination at low temperatvires . 



By choosing a n-umber of leaves of similar size and hence 

 thickness, it is possible to repeat the experiment of the previous 

 Figure 3 at various wavelengths of measuring light and to obtain 



