Electrometric and Other Studies of Cytochrome c 383 



Ehreoberg and Theorell {Acta chem. Scand. 9, 1193, 1955) had shown that if the 

 peptide chain of the 'core' were made into an a-helix cither the imidazole or the 

 c-NHo group could be made to coordinate with the central hacm iron atom, but not 

 both at the same time. However, building a suitable model it could readily be shown 

 that if the intervening chain of 4 amino acids were extended, these two groups could 

 indeed be made to occupy coordination positions 5 and 6 of the iron atom, above 

 and below the plane of the haem. 



A study of the ultracentrifugal sedimentation characteristics of the 'core' indicated 

 that although the 'core' was probably a small polymer at intermediate pH values, at 

 those alkaline pH values at which its spectrum maximally approached that of native 

 cytochrome c the 'core' was a monomer and the haemochrome must necessarily 

 have been intramolecular, rather than intermolecular as could have been the case for 

 a polymer. 



These studies (Biochem. J. 71, 559, 1959) have led us to conclude that: (a) the 

 haemochrome-forming groups in cytochrome c are probably the e-amino group 

 of the lysine in position 3 and the imidazole group of the histidine in position 8 of 

 the amino-acid sequence of the pepsin-digested cytochrome c 'core' (see Paleus' 

 paper); (b) the properties particular to the haemochrome of native cytochrome c, 

 as contrasted with those of a normal chemical haemochrome, appear to be 

 determined by the effect of the entire protein in its native configuration on the iron- 

 ligand bonds, rather than by the particular chemical groups involved in the haemo- 

 chrome of cytochrome c. 



The Amino-acid Sequence in Horse-heart Cytochrome c 



By E. Margoliash and R. Hill (Utah) 



Margoll«lSh: I should like to report on two lines of work dealing with the amino-acid 

 sequence of horse-heart cytochrome c. 



(i) Leucine aininopeptidase digestion of cytochrome c. Using a 20 % molar ratio 

 of a highly purified hog kidney leucine aminopeptidase to cytochrome c, the liberated 

 amino acids were separated by dialysis and the remaining partly digested cytochrome 

 c isolated by column chromatography on Amberlite IRC-50. Either directly or after 

 suitable acid hydrolysis both fractions were analysed on an automatic amino-acid 

 analyser. 



It was shown that about 30 amino acids are liberated after 20 hr of digestion from 

 the N-terminal sequence of cytochrome c, and that the liberated amino acids as well 

 as those remaining in the partly-digested protein add up rather accurately to the 

 composition of the original cytochrome c. These 30 residues contain the single 

 tryptophane, one of the two arginines, one of the three histidines, one of the three 

 valines and one of the four prolines in the entire protein. In addition two serines, 

 which have previously not been found in horse-heart cytochrome c were also liberated. 

 Since practically the entire haem was found in the remaining partly digested cytochrome 

 c, the haem must be located at least 30 amino acids away from the N-terminal end of 

 the peptide chain. 



(ii) Amino-acid sequence of a chymotrypsin-digested cytochrome c 'core\ Using a 

 partial digestion with chymotrypsin it was possible to isolate a chymotryptic 'core' 

 of horse-heart cytochrome c, by column chromatography on Amberlite IRC-50 

 followed by high voltage paper electrophoresis. This peptide contained 42 amino 

 acids. N-terminal and C-terminal residues were determined by reaction with fluoro- 

 dinitrobenzene and carboxypeptidase digestion, respectively. The chymotryptic 'core' 

 was digested with trypsin; the peptides formed were isolated by various combinations 

 of paper electrophoresis and paper chromatography and their composition deter- 

 mined using an automatic amino-acid analyser. 



A partial sequence thus established for the chymotryptic 'core' showed some 

 unusual features. Towards the C-terminal end there was a remarkable concentra- 

 tion of aromatic and long-chain aliphatic amino acids, enclosing a single glutamic 



