324 D. B. MoRELL, J. Barrett, P. Clezy and R. Lemberg 



cryptoporphyrin a derived from ox heart is of the order 5-10% of the 

 porphyrin a content. The lower values are probably the more correct since 

 the spectrophotometric analysis would tend to measure traces of non- 

 cryptoporphyrin a material (e.g. haematoporphyrin derived from proto- 

 porphyrin) as cryptoporphyrin a. Since it is unlikely that the ratio of concen- 

 tration of cytochrome a^ to that of cytochrome a is less than 1:3, the concen- 

 tration of cryptohaematin a in ox heart is too low for it to be the prosthetic 

 group of cytochrome a^. We thus conclude that haem a is the prosthetic 

 group of both cytochromes a and a^. 



THE PROSTHETIC GROUP OF CYTOCHROME a^ 



Several workers have shown that the pyridine haemochrome derived from 

 cytochrome a^ of various bacteria has an a band at 587 m/<, the same as 

 haemochrome a. We have considerably purified the porphyrin derived from 

 cytochrome a^ o^ Proteus vulgaris and have obtained a product (after separa- 

 tion from chlorin a^ with absorption bands identical with the bands of 

 porphyrin a from ox heart. The ratio of bands III/IV was 2-2 close to 2-4 

 for the ox heart porphyrin a. 



QUANTITATIVE MEASUREMENT OF HAEMIN a AND 

 PROTOHAEMIN IN TISSUES 



We have used two methods. (1) Extraction of the haemins, conversion to 

 the porphyrin and measurement of the protoporphyrin obtained by extraction 

 from ether into 4 % HCl and of porphyrin a obtained by extraction into 20 % 

 HCl. This method requires a minimum of about 0- 1 mg porphyrin a in the 

 tissue analysed, equivalent to 5 g wet weight of ox heart (Lemberg et al., 

 1955; 1956). For tissues other than heart muscle porphyrin a of lesser 

 spectroscopic purity is obtained and the error of determination correspond- 

 ingly increased. (2) Extraction of the haemins followed by spectrophoto- 

 metric analysis of the mixture of protohaemochrome and haemochrome a 

 derived from these haemins. This method is more sensitive and has, for 

 instance, been useful in the determination of the haem a and protohaem 

 concentration in rat tissues where the rate of uptake of ^^Fe in these haems 

 was being studied (Morell and Basser, unpublished; Lemberg and Benson, 

 1959). A disadvantage of this method is the difficulty of ensuring complete 

 formation of the haemochromes. To avoid interference from cloudiness 

 caused by lipids undissolved by the pyridine-alkali up to 15% by volume of 

 ether is often necessary. The presence of this ether makes the complete 

 reduction of the ferrihaemochrome by dithionite more difficult. 



The haemins from most tissues studied are readily extracted by acid acetone. 

 Yeasts {Saccharomyces cerevisiae, Torulopsis utilis) give some protohaemin 

 by this treatment but very little haemin a in relation to their cytochrome 

 a -\- a.^ content. It was found, however, that preliminary lysis with pyridine 



