440 R. W. ESTABROOK 



example, at 522 niju we are able to resolve absorption bands which are 

 approximately 10 A apart. Another point of interest is the fact that one 

 obtains as great an intensification of the associated absorption bands in the 

 visible region around 570-580 m/u as can be obtained at 350 mju or 445 m/^. 

 That is, there is a fifteen to twenty fold intensification of all absorption bands 

 throughout the spectrum. 



CYTOCHROMES OF THE c-TYPE 



Cytochrome c of Heart Muscle 



Figure 4 shows the type of spectrum one obtains (Estabrook, 1956) with 

 cytochrome c purified from horse heart (Note 2). The sample is reduced. 



400 430 460 490 520 550 580 



Fig. 4. A comparison of the spectral properties of heart muscle cytochrome f at 

 room temperature and at — 190"C. Purified cytochrome c was diluted in 01 m 

 phosphate buffer, pH 7-4, a few crystals of Na^S204 were added, and then an 

 equal volume of glycerol. The reference cuvette contained a mixture of an equal 

 volume of glycerol and 0- 1 m phosphate buffer. The sample and reference cells 

 were cooled in liquid nitrogen, warmed to induce devitrification of the glycerol 

 mixtures, and recooled in liquid nitrogen "(Condition II). Optical path of the 

 cuvettes was 1 mm. 



in this case, by sodium dithionite although identical results are obtained if 

 the cytochrome c is reduced enzymically. The spectrum of the same sample 

 of reduced cytochrome c, as obtained at liquid nitrogen temperature, has 

 been included. One of the first differences observed is that the a-band of 

 reduced cytochrome c, which at room temperature looks relatively sym- 

 metrical, splits into three absorption bands (Note 3) which we have termed 

 <^ai' <^"a2' ^aa- The jS-band shows an even more complex structure and by 

 precise spectral analysis one sees nine absorption bands. In addition one 

 sees an intensification of the light absorption by these a- and /5-bands of five 

 to six fold. In contrast, the Soret band does not appear as greatly intensified. 

 In addition one sees no clear splitting of the Soret band, indeed all that is 



