382 Discussion 



PosTGATE, J. R. (1956). J. gen. Microbiol. 14, 545. 



Rawlinson, W. a. (1938). Aust. J. exp. Biol. med. Sci. 16, 303. 



RODKEY, F. L. & Ball, E. G. (1950). J. biol. Cliein. 182, 17. 



Shack, J. & Clark, W. M. (1947). /. biol. Chem. 171, 143. 



Slater, E. C. (1958). Advanc. Enzymol. 20, 147. 



Takahashi, K., Titani, K,, Furuno, K., Ishikura, H. & Minakami, S. (1958). J. Biochem. 



Tokyo 45, 375. 

 Theorell, H. (1932). Biochem. Z. 252, 1. 



Theorell, H. & Akeson, a. (1941). J. Amer. chem. Soc. 63, 1804. 

 Theorell, H. &. Akeson, A. (1955). Ann. Acad. Sclent. Fennicae All, 60, 309. 

 ToEUF, G. DE. (1937). J. chem. Phys. 34, 740. 

 Tsou, C. L. (1951a), Biochem. J. 49, 362. 

 Tsou, C. L. (1951b). Biochem. J. 49, 367. 

 Yamanaka, T., Mizushima, H., Nozaki, M., Horio, T. & Okunukj, K. (1959). J. Biochem. 



Tokyo 46, 121. 



DISCUSSION 



Protein Conjiguiation and Linkage to the Prosthetic Group in Cytochrome c 

 Studies of the Haeraochrorae-forming Groups in Cytochrome c 



By E. Margoliash (Utah) 



Margoliash: For all the typical haemochrome properties there is no sharp breaking 

 point between native cytochrome c on the one hand and the pepsin digested cytochrome 

 c 'core', which can be considered as the most degraded form of the cytochrome c haemo- 

 chrome, on the other. Quite the contrary, by isolating by column chromatography 

 a series of cytochromes c showing increasing degrees of denaturation it could be 

 shown that all the haemochrome properties vary gradually with the degree of 

 denaturation. These fractions of denatured cytochrome c were defined by their 

 chromatographic properties on Amberlite IRC-50, the more protein being de- 

 natured the larger the number of e-NHz groups of lysine available to the resin and for 

 reaction with a reagent such as l-fluoro-2-nitro-4-benzene sulphonate. 



Native cytochrome c was at one end of the scale with maximal enzymic activity in 

 the succinic oxidase and cytochrome oxidase systems, a very low rate of auto-oxidation, 

 a small per cent combination with CO at near neutral pH values, and a very wide 

 pH-range of stability of the reduced spectrum. With increasing degrees of denaturation 

 the enzymic activities became lower, the rate of auto-oxidation and the per cent 

 combination with CO increased ; the pH-range of stability of the reduced spectrum 

 became gradually smaller moving toward the alkaline side. With respect to all these 

 properties the cytochrome c 'core' is in every way identical to the most denatured 

 form of cytochrome c obtained, even though it contains only 1 1 amino acids out of 

 the full complement of 96-98 present in denatured cytochrome c. 



The cytochrome c 'core' shows on reduction the typical cytochrome c haemo- 

 chrome spectrum, but in contradistinction to native cytochrome c, the reduced 

 spectrum of which is completely unaffected by pH over a very wide range from pH 2-3 

 to pH 13, the 'core' acts like a normal chemical haemochrome. Measuring at the 

 maximum of the a band, one finds that the spectrum of the 'core' reaches the full 

 extinction of native cytochrome c only at about pH 11 after passing through 3 sets 

 of inflexions with mid-points at 5-4, 7-6 and 9-5. The addition of a large excess of 

 bases containing a- or e-NH, groups did not effect the maximal extinctions obtained 

 at pH 11, whereas the addition of bases containing only an imidazole group decreased 

 the height of the a-band to 5-8 % below that of native cytochrome c. This was in- 

 terpreted as indicating that the haemochrome-forming groups in cytochrome c 

 are not both imidazoles. We therefore investigated the possibility that the e-NHg 

 group of the lysine and the imidazole group of the histidine, both amino acids being 

 the residues adjacent to the cysteines involved in the thio-ethcr bonds holding the 

 haem to the protein, were the actual haemochrome-forming groups in cytochrome c. 



