384 Discussion 



acid, and in the centre of the sequence a concentration of basic residues (3 or 4 

 lysines and one histidine). Between these two regions was located a single proline. 



Mammalian heart cytochrome c has no known covalent bonds holding the protein 

 together, it is readily denatured by acid and alkali and although all of its acidic and 

 basic residues are titratable, only 8-9 of the e-NH, groups of its 18 lysines will react 

 rapidly, in the native protein, with a water soluble reagent such as l-fluoro-2-nitro-4- 

 benzene sulphonate. I should therefore like to propose the hypothesis that concen- 

 trations of long chain aliphatic and aromatic amino acids in specific positions provide 

 the required hydrophobic microenvironment to stabilize particular acid-base ionic 

 bonds which serve to keep the protein in its native folded configuration. What was 

 observed in the chymotryptic 'core' would presumably be one such region. 



Another consequence of this work is that the haem is attached at least 30 residues 

 away from the N-terminal end and at least 30 residues away from the C-terminal end, 

 thus putting the haem to probably within 15 residues of the centre of the peptide chain. 



Comments on the Structure of Cytochrome c 



Paleus: As to the spectrophotometric curve shown (by Margoliash) of the peptic horse 

 haemopeptide, and the effect of addition of histidine to the solution, I want to refer 

 to the results of Tuppy and Bodo (M/j. Chem. 85, 1024, 1182, 1954), as well as those 

 of Ehrenberg and Theorell {Acta chem. Scand. 9, 1193, 1955). These authors added 

 histidine (in the case of Tuppy and Bodo also a-benzoyl-histidine) to a solution of the 

 tryptic horse haemopeptide of cytochrome c (Tuppy and Bodo) and to the peptic 

 beef haemopeptide (Ehrenberg and Theorell) at pH 7-3 and 8-9 respectively. They 

 found that the extinction at 550 m// was raised to that found in native cytochrome c. 

 Margoliash, however, observed this phenomenon first at pH 11. 



As to the haem-Unkage, I want to refer to the results of Tuppy and Bodo, who were 

 able to show how the autoxidizable haemopeptide lost most of its autoxidizability on 

 adding histidine at the same time as the extinction at 550 m^ of native cytochrome c 

 was reached. Ehrenberg and Theorell showed spectrophotometrically the inability of 

 ferro- and ferric protoporphyrin at pH 7 to form any compounds with lysine or 

 glycyl-glycine. 



What does Margoliash now think is the N-terminal group of his cytochrome c 

 preparation? Is it histidine as he stated in 1955? Did the N-terminal group show up 

 at the degradation of cytochrome c with leucine aminopeptidase, the experiment 

 which he has shown us today? However, Minakimi, Titani and Ishikura (/. Biochem. 

 Tokyo, 45, 341, 1958) could not degrade cytochrome c with leucine aminopeptidase. 

 Their results do not correspond to yours. 



As to your opinion of the electrostatic holding together of the cytochrome c molecule 

 it may be rather important. I also want here to mention that one can abolish the 

 capacity of the enzyme for electron-transfer by, e.g. acetylation of the e-NHo-lysine 

 group, but not by guanidation, as shown by Minakami and co-workers. 



Margoliash: I do not think that the results of Tuppy and Bodo (who found that adding 

 a large excess of a-benzoylhistidine to the tryptic core of cytochrome c resulted in a 

 considerable increase in the reduced a-band extinction) conflict with our own results. 

 If I remember correctly, Tuppy and Bodo obtained an increase of the a-band extinction 

 to about 80-90% of that of native cytochrome c at somewhat alkaline pH. This is 

 very similar to our own results with imidazole and the pepsin digested cytochrome c 

 'core', and does not influence the fact that no externally added bases are required to 

 obtain a spectrum virtually identical to that of native cytochrome c, at pH 11, with 

 this 'core'. You asked whether we managed to establish an N-terminal amino acid 

 for cytochrome c with leucine aminopeptidase. No, this has not been done. Leucine 

 aminopeptidase docs not produce an appreciable digestion at too low a concentration. 

 On the other hand, at concentrations that will work, the rate of digestion is so rapid 

 that it is not easy to establish by a kinetic study which is the first amino acid liberated. 

 There is of course the added difliculty that if the first peptide bond to be split is 

 broken slowly as compared to the next few bonds along the chain, all these amino 



