69 



Britton Chance and Walter D, Bonner, Jr. 



EXPEEUMENTAL METHODS 



A Dewar flask is attached onto the emergent light fitting of 

 the doulJle-heara spectrophotoaeter (9, 10). Tiie one inch photo -rnul- 

 ti-olier is attached directlir to the housing of the Dewar flask. 

 Since this Dewar has right-angle windows, side illumination is 

 readily obtained from either a tvingsten lemp (680 va\i illumination) 

 with appropriate interference filter or a mercury arc (^3^ "Ua 

 illxomination) with a multielement filter (Eppendorf). The cuvette 

 is held in the pictxire and contains an aluminiom block with a pair 

 of lucite plates between which the leaf is gently pressed. A 

 smsill mirror serves to reflect the actinic light upon the leaf, 

 out of the way of the measuring light. All other aspects of the 

 double-beam spectrophotometer are as in previous communications 

 (11). The levels of actinic illumination are low; since cyto- 

 chrome f responds as a "quantum counter", high levels are not 

 required to cause maximal oxidation as they are at room tempera- 

 ture. We are not reporting quantum efficiencies for cytochrome 

 f oxidation in this paper and therefore light intensities are 

 reported in arbitrary \inits. The values are about 10"^^ to 10"^/ 

 Einsteins/cm^/sec . 



\^ea k36 m\x excitation is employed, the rate of cytochrome f 

 oxidation is slower than with 680 m^i excitation, as indicated in 

 the Figures below. However, the extent of the reaction is the 

 same, since the reaction is essentially irreversible. 



Absorbancy enhancement . 



With cytochrome c the absorbancy increment in the oxidized 

 minus reduced spectra at '^9 rap is if. 5 times greater. Presumably, 

 such values apply to leaves, but detailed controls are necessary 

 to ensure this. This factor is not known for P700. For the 

 purposes of this paper, we are emphasizing the ratio of the rates 

 at 555 and 705 m\x. This comparison may be made by assuming the 

 same enhancement for the two wavelengths without the need for 

 its absolute value. 



Effect of the measuring light . 



At low temperatures, cytochrome f becomes effectively a "quan- 

 tum counter" and therefore, prior illTomination with undue inten- 

 sities of excitation light will vitiate the desired response. 

 We have, however, illustrated the effect of a high level of 

 measuring light intensity in Figure 2 . In this illustration, the 

 measuring light is that obtainable from a 3 mn slit of the Bausch 



