VOL. 12 (1953) 



CO COMPOUNDS OF RESPIRATORY ENZYMES 



291 



two suspensions of respiring cells ot equal concentration. Cuvette A is taken to be the 

 reference cell and the photocurrent obtained upon illuminating that cuvette is main- 

 tained at a predetermined level by an automatic gain control circuit that receives its 

 signal from the contacts of the first demodulator and adjusts the dynode voltage of the 

 photomultiplier to the appropriate value for constant photocurrent regardless of the 

 intensity of the light illuminating the cuvette, the transmission through the sample, or 

 the sensitivity of the photosurface. This constitutes the "100% transmission" signal. 

 When cuvette B is illuminated, a signal representing the actual transmission is received 

 from the first demodulator and is measured by a second demodulator. In order to record 

 optical densities, the percent transmission is converted into logarithms by a segmented 

 logarithmic characteristic consisting of ten diodes^^. 29. The optical density values are 

 then plotted by a servo recorder (Leeds and Northrup Speedomax) . In order to have a 

 linear wavelength scale on the chart, an appropriately loaded potentiometer operates in 

 the feedback circuit of the chart drive servomechanism^^. 



Ropid Recording Spectrophotometer 



Tungsten or 

 high pressure 

 Hg arc /^ 

 lamp ^— ^ 



Monochromator 

 (quartz prism) 



Cuvettes 



Electron 



multiplier 



ptiototube 



First 

 Oemodulotor 

 and g c 



Second 

 Demodulator 



Logarithmic 

 Converter 



Synchronized Chart 

 with linear wovelength scole 



Optical Density 



Recorder 



Fig. I . A schematic diagram of the operation of a spectrophotometer suitable for recording the spectra 

 of the respiratory pigments of cell suspensions and tissue homogenates. age represents "automatic 



gain control" (MD-25). 



The apparatus has a noise level of --^ i •lO"'^ in optical density and operates with 

 a spectral interval of 2 m/x or less when the cuvettes are filled with a turbid suspension 

 of respiring cells. The spectrum is plotted at the rate of a few millimicrons per second. 



In studies with turbid cell suspensions, it is important to gather both the transmit- 

 ted and the scattered light from the cell suspension in order to obtain adequate sensitivity. 

 This we accomplish by placing the phototube near the cell suspension and thereby avoid 

 the lens and prism as were used earlier by Warburg and Christian^. 



In actual use, a "base-line" is plotted with cuvettes A and B hlled with equal con- 

 centrations of oxidized cells. Then the substrate is added to the cell suspension in cuvette 

 B so that the oxygen is consumed and the absorption bands of the reduced cytochromes 

 are recorded. Next the substrate is added to cuvette A and a second base-line is drawn. 

 CO is finally bubbled through cuvette B to form the CO-reduced compound and the 

 spectrum of the CO compound is plotted. (For further details see reference 9.) 



Thus this apparatus is especially useful for plotting difference spectra, for example, 



References p. 2gyl2g8. 



