STRUCTURE AND P^UNCTION OF SOME ENZYMES 



139 



creased to 0.43 per cent. "We have some rea- transformed reversibly one into another, 



sons to think that these preparations are 

 definitely pure. The analytical data do not 

 differ very much from those of any common 

 protein (52.5 per cent C, 7.76 per cent H, 

 16.66 per cent N, 1.5 per cent S = 6 atoms 

 per mol., 0.43 per cent Fe = 1 atom per 

 mol. ) . The molecular weight is 13,000. 



giving dissociation curves, which enabled us 

 to measure the dissociation constants spec- 

 trophotometrically. Ferrocytochrome-c, on 

 the contrary, at any pH value shows the 

 typical, two-banded spectrum, 550 and 520 

 m^j. Some of the results with ferricyto- 

 chrome are given in Table 1. 



TABLE 1 



Cytochrome-c shows some striking fea- 

 tures. It gives no addition compounds with 

 oxygen, carbon monoxide, hydrogen cya- 

 nide, fluoride, or azide around the neutral 

 point, which indicates that the iron atom 

 of the hemin is blocked by some strongly 

 linked groups of the cytochrome molecule 

 itself. Furthermore, there is another strong 

 linkage between the protein part and the 

 porphyrin, because unlike hemoglobin, cyto- 

 chrome-c cannot be split up into hemin and 

 protein by means of acetone and hydro- 

 chloric acid. As a matter of fact, no less 

 drastic treatment than hydrobromic acid in 

 glacial acetic acid will liberate the porphy- 

 rin from the residues of the protein com- 

 ponent. Now cytochrome-c has an amazing 

 property that has made it possible for us to 

 find out a great deal about its constitution. 

 It is probably the most "pH-stable" pro- 

 tein yet known. Neither normal hydro- 

 chloric acid nor normal sodium hydroxide 

 destroys it, at least for a reasonably short 

 time at room temperature. Therefore, it 

 has been possible to study its spectral be- 

 havior, its magnetic susceptibility, and its 

 formation of compounds with CO, HON, 

 and fluoride in acid or alkaline solutions. 



It was found that ferricytochrome-c forms 

 not less than five spectroscopically different 

 compounds, depending on the pH of the 

 solutions. By changing the pH they are 



The spectral behavior of ferricytochrome 

 may be explained on the assumption that 

 the hemochromogen-forming basic groups 

 are primary amino groups, probably be- 

 longing to the protein component. The five 

 different spectral types then should corre- 

 spond to the following constitutional types : 



At physiological hydrogen-ion concentra- 

 tions ferricytochrome-c occurs exclusively 

 as type III, which does not give any com- 

 pounds with azide, fluoride, or cyanide. 

 Under the same conditions ferrocyto- 

 chrome-c gives no addition compounds with 

 oxygen or carbon monoxide. This means 

 that the six valencies of the iron atom are 

 here so firmly occupied by the six nitrogen- 

 atoms that none of the compounds men- 

 tioned can replace any of the nitrogen- 

 atoms. There may also be a sort of steric 

 hindrance, if we postulate the flat, disc- 

 shaped haem molecule to be inserted into a 

 crevice of the protein molecule: 



It will be seen from the formula of cyto- 

 chrome-c, that the haem is connected with 

 the protein in a twofold manner, the iron- 

 atom by means of amino groups and the 

 a-atoms of the side chains in the 2- and 

 4-positions by means of very stable ether 

 linkages.^ These latter linkages are the 

 cause of the high stability of cytochrome-c 



2 Possibly thio-ether Linkages with cysteine sul- 

 phur (Theorell). 



