Electronwtric and Other Studies of Cytochrome c 385 



acids will be liberated practically simultaneously, making it impossible to decide 

 which is the N-terminal. 

 Williams: I believe that the observations of Margoliash can also be interpreted in terms 

 of Theorell's original suggestion that the cytochrome was a di-imidazole complex. 

 Rather than go into detail here it is proposed to submit a full analysis of these experi- 

 ments elsewhere (see also, this volume, p. 141). 



Structure and General Properties of Cytochrome c 



Comparative Properties of Cytochrome c from Yeast and Heart Muscle 



By J. McD. Armstrong, J. H. Coates and R. K. Morton (Adelaide) 



Morton: This paper relates particularly to the contribution by Henderson and Rawlinson 

 (this volume, p. 369) and describes studies briefly reported elsewhere (see Armstrong, 

 Coates and Morton, 1958; Morton, 1958). 



Air-dried baker's yeast (as used for isolation of crystalline cytochrome b.^; see 

 Appleby and Morton, 1954, 1959) was extracted with m NaCl for about 6 hr at room 

 temperature (about 22°C) or for 30 min at 55-60°C. The supernatant obtained on 

 centrifuging was dQuted with three volumes of water and adjusted to pH 5-7. The 

 cytochrome c in the extract was absorbed batchwise onto resin (IRC-50(Na+)), and 

 after washing the resin, the cytochrome c was eluted either with 0-8 m NaCl in 0-1 

 m sodium phosphate pH 7-8, or 40% saturated ammonium sulphate pH 8-5-90. The 

 cytochrome c was purified by chromatography on a column of the same resin until the 

 ratio of E549 tsxfij^^n ^l<^ was constant (1-05-1 -12) for the eluted cytochrome. 



Electrophoresis by the moving boundary method was carried out in buffers of 

 ionic strength of 0-2, at 11 pH values from pH4-18 to pH 1007, At all pH values 

 only one protein component was detected, although some asymmetry of the boundary 

 was apparent. Only microheterogeneity could be detected by reversal of polarity 

 after prolonged electrophoresis at pH 9-93, which is close to the iso-electric point of 

 the cytochrome. 



Figure 1 shows the pH-mobility plot for the yeast cytochrome c in comparison with 

 the plot obtained by Theorell and Akeson (1941) and by Tint and Reiss (1950) for 

 ox-heart cytochrome c. The iso-electric point for yeast cytochrome c is pH 9-85 ± 0-05 

 under our conditions, as compared with pH 10-6 for the ox-heart cytochrome c. 



Sedimentation at 60,000 rev/min in a Spinco analytical ultracentrifuge showed only 

 a single component. Preliminary studies using the Archibald approach-to-equilibrium 

 procedure indicate a weight/mole of approximately 15,000 g, in reasonable agreement 

 with the minimum weight/mole of 14,700 g estimated from the iron content of 0-38 %. 



The Eq value at pH 6-4 was determined as +0-282 V by Henderson and Rawlinson 

 (see this volume, p. 371), as compared with +0-255 V for ox-heart cytochrome c under 

 similar conditions. 



Table 1 is a summary of the comparative properties of yeast and heart-muscle 

 cytochrome c (see also Morton, 1958). It is apparent that the two proteins are not 

 identical. The lower iso-electric point for yeast cytochrome c is consistent with the 

 lower lysine content (15-16 and 18 residues/mole in yeast and heart-muscle cytochrome 

 c respectively) reported by Nunnikhoven (1958). 



It is noteworthy that only one protein component was detected in these studies. 

 Nunnikhoven (1958) extracted cytochrome c from yeast by Keilin's (1930) procedure, 

 concentrated the extract on resin, and fractionated with ammonium sulphate after 

 acidification with trichloroacetic acid (TCA). By electrophoresis at pH 8-3, two 

 haemoprotein components were detected (containing 0-36 and 0-42% of iron for the 

 faster and slower component respectively). Tsou (1956) obtained yeast cytochrome c 

 by autolysis of dried yeast in m NaCl, concentrated it on a synthetic zeolite, eluted 

 with 72 % saturated ammonium sulphate, and precipitated the cytochrome with TCA. 

 Two haemoprotein components were detected, capable of separation by electrophoresis 

 at pH 6-3. The major component had an iron content of 0-43 %. 



