292 



B. CHANCE, L. SMITH, L. CASTOR 



VOL. 12 (1953) 



+ .030 



_ +.020 



+.010 



the spectrum representing the difference between the reduced and oxidized forms of the 



respiratory pigments of 5. albus illustrated by the trace labelled "reduced" in Fig. 2. 



And the trace labelled "reduced + CO" represents 



the difference between the CO compounds and the 



reduced forms of the respiratory pigments. In this 



case one observes a distinct peak at 416 mju. and a 



trough at 432 m/x, as contrasted to the peak and 



trough of the corresponding spectrum for the yeast 



cells which lie at 430 and 445 mju, respectively. 



Similar studies can be carried out for S. albus 

 in the visible region of the spectrum and peaks at 

 535 and 570 mjLt are reported by Smith^^. 



Fig. 2 when compared with Fig. 3B of refer- 

 ence 9 shows that cultures of 5. albus may be 

 treated so as to increase considerably their relative 

 content of this CO-binding pigment. 



E 

 « 



S -.010 



-.020 



o -.030 



o. 

 O 



Fig. 2. The absorption difference spec- 

 tra for a culture oi Staphylococcus albus 

 obtained by means of the apparatus of 

 Fig. I. The trace labelled "reduced" 

 represents the differences between the 

 reduced and oxidized cytochromes and 

 the trace labeled "reduced -|- CO" 

 represents the difference between the 

 CO compound and the reduced form. 

 Similar data can readily be obtained 

 in the visible region (0-37). 



Photodissociation difference spectra 



Because their experimental conditions were in- 

 appropriate, Keilin and Hartree were not able to 

 demonstrate the photochemical dissociation of the 

 CO compound of cytochrome a^. We have recently 

 been able to accomplish this by a differential 

 spectrophotometric method that is suitable for the 

 observation of changes in absorption due to the 

 photodissociation reaction within the respiring cell. 



This experiment is considerably more difficult than that carried out by Bucher and 

 Negelein on clear solutions of hemoglobin and myoglobin carbon monoxide. It is not 

 possible to use here the favorable optical geometry that they used — -a short optical path 

 for the photodisiociating light and a long path for the measuring of light. With turbid 

 cell suspensions, we require a fairly large surface area of the suspension near the meas- 

 uring photosurface (see p. 291, 2nd paragraph under Fig. i). Thus we have used a square 

 cuvette in which the photodissociating and the measuring paths are both equal to one cm. 



Another novel feature of the method that we use is the ability to vary the measuring 

 wavelength and thereby to obtain a "photodissociation difference spectrum"* of the CO 

 compound. Since the turbid suspensions scatter photodissociating light of very high inten- 

 sity in the direction of the measuring phototube, our method has three features that avoid 

 interference with the spectrophotometric measurement by the photodissociating light. 



* In order to distinguish between the three types of spectra of the CO compounds that are dis- 

 cussed in this paper, it is useful to define the three terms: 



Absorption difference spectrum. This is a spectrum representing the change of light absorption 

 caused by a chemical change of the pigment, for example, from oxidized to reduced (a reduced-ox- 

 idized spectrum), or from reduced to the CO compound (a CO-reduced spectrum). 



Photodissociation difference spectrum. This is a spectrum representing the change of light absorp- 

 tion caused by a photochemical reaction, for example, the photochemical dissociation of a CO com- 

 pound of a reduced cytochrome in which case a CO-reduced spectrum is obtained. 



Photochemical action spectrum. This is an absolute (not difference) spectrum. The ordinates are 

 inversely proportional to the quantum intensity required at each wavelength to produce a given 

 rate of photochemical decomposition of the CO compound. 



References p. 2g7l2()8. 



