150 A SYMPOSIUM ON RESPIRATORY ENZYMES 



described a simpler method of preparation involving a trichloracetic 

 acid extraction of the ground muscle, ammonium sulfate fractiona- 

 tion, and trichloracetic precipitation. Both groups of workers ob- 

 tained a product which had the same absorption bands as the 

 cytochrome c of the intact or phosphate-extracted muscle, and 

 which contained 0.34 per cent iron, resisted further fractionation, 

 and was therefore considered pure. Later Theorell and Akesson (6) 

 obtained by electrophoretic means a product containing as high as 

 0.43 per cent iron. It would be of interest to know whether this 

 product displayed an equal increase in catalytic activity over the 

 0.34 per cent iron product. 



Zeile and Renter (7) calculated a molecular weight of 18,000 for 

 cytochrome c on the basis of its hemin content. Theorell (4), from 

 a study of its diffusion and sedimentation, determined a molecular 

 weight of 16,500. The iron content of 0.34 per cent also yields an 

 equivalent weight of 16,500. The isoelectric point of cytochrome c 

 is approximately at pH 9.8 (4). 



Ferric cytochrome c is readily reduced by a variety of agents, 

 such as hydrosulfite, ascorbic acid, cysteine, adrenalin, hydroqui- 

 none, p-phenylenediamine, and many leuco dyes, as well as by 

 certain physiological reducing systems to be discussed later. 



Ferro -cytochrome c is essentially non-autoxidizable in neutral 

 solution, the slow rate being largely inhibited by small amounts of 

 cyanide (8), indicating heavy metal catalysis. It is readily oxidized 

 by ferricyanide and aerobically by oxidase preparations. Below pH 

 4.0 and above 11.0 the spectrum changes and the substance becomes 

 autoxidizable. 



A neutral solution of cytochrome c can be boiled, and upon cooling 

 the original spectrum and catalytic properties return. Cytochrome c 

 is likewise stable to dilute acid. It is stable to 0.1 normal potassium 

 hydroxide, but 1.0 normal alkali produces an irreversible change in 

 the spectrum. The substance is then autoxidizable and forms a light- 

 sensitive carbon monoxide compound (9). 



According to Keilin (10), cytochrome c does not appear to com- 

 bine with hydrogen sulfide, hydrogen cyanide, sodium azide, or 

 hydroxylamine, nor, according to Stern (11), with carbylamine. 

 Ferri-cytochrome c does, however, form a compound with nitric 

 oxide (12). Keilin (13) was unable to detect any change in the 

 spectrum of the c component in the presence of carbon monoxide 

 except in solutions whose pH was above 13.0. Altschul and Hogness 

 (14), using an accurate photoelectric spectrophotometer (15) with a 



