71 



Britton Chance and Walter D, Bonner, Jr. 



and Lonib monochromators. Upon the moment of illumination of the 

 sample, a steady state deflection of the two traces is observed 

 which increases when the actinic light (680 mu) is turned on. It 

 is seen that the measviring light intensity is adequate to cause 

 an appreciable rate of oxidation of cytochrome f and roughly half 

 of the cytochrome f has been oxidized by the time the actinic 

 light is t\irned on. 



A criterion of satisfactory operation may be taken as the ra- 

 tio of the response time of the spectrophotometer (0.3 sec for 10 

 to 90 per cent in this case) to the time for the measuring light 

 to cause an arbitrary oxidation of cytochrome f (lO per cent is 

 used here). This ratio, M, is over kO here and may be useful in 

 comparing different spectrophotometers. 



With the actinic illumination employed here, a response time 

 of 1 sec (10 to 90 per cent) is adequate in order to diminish the 

 oxidation of cytochrome f prior to illumination. VJe have reduced 

 the spectral interval from 3 to about 1 mji. In most of the 

 traces which we report here, the effect of the meas\iring light 

 prior to illumination with actinic light is negligible; the ratio 

 M is over 25, even when using 7OO mij. as the measuring light. 



The recording is usually made with double traces at different 

 gains and at different response speeds. Thus, the rapidly re- 

 sponding trace ( showing an upvrard deflection) is at a lower gain 

 and at a higher response speed (l sec), while the downward de- 

 flecting trace is at a higher gain an a lower response speed (2- 

 3 sec). However, the respective upward and downward deflections 

 of both traces correspond to a decrease of absorbancy at 555 with 

 reference to 5^0 m^. 



In these studies in which the absorbancy changes due to P70O 

 are measured, certain controls and precautions are observed 

 to insure that fluorescence changes are not causing artifac- 

 tual responses. First, it should be pointed out that the 

 observations of Butler (12) on the fluorescence of chloro- 

 phyll show that fluorescence enhancement of approximately 20 

 per cent caused by light in the region of 620 myi is decreased 

 considerably by light of wavelengths of TOO mn. Therefore, a 

 fluorescence artifact would be in the opposite direction from 

 the absorbancy decrease observed; i.e., 705 ^P- illumination 

 decreases the long wave fluorescence of chlorophyll relative 

 to that of 635 niP-. Thus, with the double-beam spectrophoto- 

 metric technique, the absorbancy change woi^ld not be confused 

 with the fluorescence change, as they are in opposite direc- 

 tions. Secondly, the abil5.ty to record the kinetics on a fast 



