350 VIII. HEMATIN ENZYMES, I. CYTOCHROME SYSTEM 



ing hemoglobin and other impurities (960,2334,3768). In the resulting solu- 

 tion the absorption of ferrocytochrome c is determined spectrophotomet- 

 rically. Finally cytochrome c may be extracted, and the difference of the 

 absorption of ferrocytochrome c and ferricytochrome c in the extract may be 

 determined spectrophotometrically {2179). This method, which is preferable 

 to the second, uses a kidney preparation containing both cytochrome oxidase 

 and dehydrogenases in excess. Without substrate ferrocytochrome is thus 

 oxidized to ferricytochrome while in the presence of substrate and of cyanide 

 to poison the oxidase, cytochrome c is fully reduced. 



3.3.2. Nature of the Active Center of Cytochrome c and Its 

 Linkage to Protein. By action of sulfur dioxide on cytochrome c, 

 Hill and Keilin (1284) had obtained a water-soluble, ether-insoluble 

 "porphyrin c." By treatmer^ with hydrobromic acid in acetic acid 

 this yielded hematoporphyrin, which Zeile and Renter {3171) trans- 

 formed to mesoporphyrin IX. The arrangement of the side chains 

 on the porphin nucleus of cytochrome c is thus identical with that in 

 protoporphyrin and hemoglobin. Theorell {2768,2769) showed that 

 "porphyrin c" of Hill and Keilin still contained peptide groups. On 

 further hydrolysis it lost much amino nitrogen, but not its sulfur. 

 The new porphyrin c contained two sulfur atoms in thioether linkage 

 and two primary amino groups. Two of the carboxylic acid groups 

 (those of the propionic acid side chains) were found to have a pK 5.7, 

 while two additional amino acid carboxyl groups of the cysteine were 

 only weakly acid (pK 9.22). The compound split off by hydrobromic 



NHz 

 S CH2CHCO2H 



CHS— CH2CHCO2H 



Fig. 1. Porphyrin c from cytochrome c. 



acid in acetic acid was identified as L-cystine {2770). The position 

 of the hemochrome bands of cytochrome c shows that it contains no 

 vinyl side chains (c/. 621). Theorell concluded from these results 



