294 



B. CHANCE, L. SMITH, L. CASTOR 



VOL. 12 (1953) 



causing photodissociation are operated on well-filtered direct current, there is no alter- 

 nating component of the yellow light that leaks through the filters. 



By means of these three design factors, the filtered light from a 100 watt Na or Hg 

 lamp a few inches from the sample cell causes no deflection of the trace of the spectro- 

 photometer. The only detectable effect of the light leakage is an increase in the shot 

 noise output of the photoelectric circuit. 



A t5^pical record of the photodissociation and recombination of the CO compound 

 in bakers' yeast cells is shown in Fig. 5. Starting with the steady-state oxidized yeast 

 cells in the presence of alcohol, reduction of cytochrome a^ causes the abrupt increase 

 of optical density at 445 m^u. (with respect to 480 m/x) as indicated by the downward 

 sweep of the trace. Illumination of the cells with the Na arc at this time results in no 



Reduced 



{ 2.2 mjj slit) 



430- = 



480 m;j = 



(2.8m;i slit)= 



_ 437- = 

 ^480m;j= 

 (2.5mjj slit)" 



l_ +.0I0' 



Fig. 5. An example of the measurement of a photodissociation difference spectrum of the cytochrome 

 a.,-CO compound of bakers' yeast cells with the apparatus of Fig. 4. The points "on" and "otf" re- 

 present the moments at which the photodissociating light is turned on and off. Illuminating light 



is 589 mfi. (25° C) (Expt. i45e). 



deflection of the trace. When reduction is complete and the cells are substantially anae- 

 robic, a solution of CO is added to give a final concentration of 5.5 fxM, causing the for- 

 mation of the cytochrome a^-CO compound. IHumination of the cefls now causes the 

 dissociation of the CO compound while darkness allows its reformation, the latter change 

 corresponding to a decrease of optical density and to a trough in the difference spectrum. 

 If now the wavelength is shifted to 430 m/x, illumination causes the opposite sign of op- 

 tical density change to occur, corresponding to a peak in the difference spectrum. And 

 if a wavelength of 437 m/x is used no change at all occurs; this is an isosbestic point be- 

 tween the reduced and CO-reduced spectra pro- 

 viding a good control against possible artifacts. 



These deflections, plotted as a function of wave- 

 length, form a "difference spectrum" that represents 

 the differences between the absorption of the CO com- 

 pound and the reduced form of cytochrome a^. The 

 peak of this difference spectrum would be expected 

 to lie very close to that of the absolute photochemi- 

 cal absorption spectrum of the respiratory enzyme at 

 430 m/x^^ because, as Keilin and Hartree already 

 have shown^^, the respiratory enzyme has many of 

 the properties of cytochrome a^. The result obtained 

 with direct methods affords a conclusive proof of 

 the identity of the respiratory enzyme of T. utilis 

 and cytochrome flg of heart muscle preparations. 



S. alhus shows a rather different pigment with a peak at 415 m/x and a trough a 

 433 m/x as shown in Fig. 6 ^^. 



References p. 2gyJ2g8. 



c 



e 



<u 



c 

 Q 



a. 

 O 



-.010 



Dork 



400 



460 



420 440 



>(m>j) 

 Fig. 6. A photodissociation difference 

 spectrum for the "CO-binding pig- 

 ment" oi Staphylococcus albus obtained 

 with the apparatus of Fig. 4 (948 c). 



