Eleclrometrk and Other Studies of Cytochrome c 377 



Recently, however, we have been successful in crystallizing the pigment 

 from a '0-31 % Fe' content sample obtained simply by the usual KeiUn and 

 Hartree procedure. The crystallization method of Hagihara, Morikawa, 

 Tagawa and Okunuki (1958) was followed except that an addition of copper 

 was made in the binding ratio of 1 g atom Cu/4 moles cytochrome c (see 

 Henderson and Rawlinson, 1960). Small crystals formed rapidly (1-2 hr) 

 and within 48 hr many rosettes were present along with crystals (Fig. 5) 

 having an appearance very similar to those depicted by Hagihara et al. 

 (1956) and described by them as leaflets. The crystals were in the reduced 

 form. The biological activity and other properties of this preparation have 

 not yet been investigated. 



Possible Linkage of Cytochrome c in vivo 



The above experiments show that a colourless protem fraction is associated 

 with M. cytochrome c extracted both with and without the use of acid, and in 

 the latter case when myoglobin and haemoglobin would appear to have been 

 completely removed. This fraction has an anodic mobility at pH 7-4 and from 

 this fact it is to be expected that some affinity exists between this material 

 and M. cytochrome c with its high iso-electric point (pH 10-5). There is 

 some evidence that the fraction is globin but from the above experiments it 

 would seem that there is still room for doubt. 



The view has been put forward previously that this material is associated 

 with cytochrome c in vivo (see Lemberg and Legge, 1949). This view was 

 based largely on the behaviour of '0-34% Fe' cytochrome c with regard to 

 such properties as heat stability and difficulty of separation except by electro- 

 phoresis. If it is in fact present in vivo it may be that the strong metal- 

 binding capacity shown above is the means of bringing about the particular 

 spatial or other arrangement — even of electron transport — which it seems is 

 necessary (see Slater, 1958) for full endogenous activity. 



^Modified'' Cytochrome c 



After separation of the main fraction of cytochrome c on a resin column 

 (Fraction I, pH 7 according to Margoliash, 1954b), there is left a band of 

 pigment at the top of the column. This material (Fraction II) may be eluted 

 with 0-5 N NH4OH. Margoliash (1954b) found it to have an increased 

 ascorbic acid oxidase activity and a lowered biological activity. Some of the 

 colourless protein fraction is, however, eluted at the same time by the 

 NH4OH. After further chromatography to remove as much as possible of 

 the latter, a relatively high ascorbic acid oxidase activity resulted (curve C, 

 Fig. 2). ^ 



The Eq value of this fraction has already been mentioned (Table 2) to be 

 some 55 mV above that of the main fraction. It also seems very likely that 

 the state of aggregation is altered, as it is seen from Fig. 6 that the value of 



