The Cytochromes of Plant Tissues 



485 



Since it was impossible to determine the osmotic concentrations of each 

 tissue used in this investigation, one standard procedure for isolation of 

 cytoplasmic inclusions was used; this procedure is outlined in Fig. 2. In 

 cases where the tissues were known to be fairly acid, 0-05 m potassium 

 bicarbonate or 0-01 m Tris (2-amino-2-hydroxymethylpropane-l :3-diol) 

 buffer, pH 7-8 were used in place of the phosphate. A more detailed description 

 of the isolation of plant particles is given by Bonner and Smith (1961). 



TEMP ABSORPTION CELL 



Fig. 1 . The absorption cell used for low temperature studies. During the optical 



measurements, the foot remains in the liquid nitrogen, thus insuring that the 



temperature of the absorption cell and its contents remains close to — 190°C. 



The use of low temperature spectrophotometry is essential for the investi- 

 gation of plant cytochromes for two reasons : (a) the relatively low concen- 

 tration of cytochrome components in plant tissues and in cell-free prepara- 

 tions derived from them, a situation where absorption intensification at low 

 temperature is almost mandatory ; and (b) the multiplicity of the cytochrome 

 b components of close absorption maxima requires the sharpening and 

 definition that is obtained at low temperatures. 



In this investigation, four types of difference spectra were used to delineate 

 the cytochromes in particulate preparations. In each case, the spectra were 

 representative of the difference in absorption between an aerated preparation 

 and a preparation treated in one of the following ways : 



1 . Substrate reduction to different steady states. 



2. Reduction with reduced diphosphopyridine nucleotide (DPNH) in the 

 presence of cyanide. 



