The Isolation, Purification and Properties of Haemin a 323 



Conversion of Porphyrins to Haemins 



In the absence of HCl the equilibrium of the system, porphyrin + Fe++ 

 ^ haemin + 2H+, strongly favours haemin formation. The reactants are 

 heated to 80°C to obtain a reasonable reaction rate. 



The whole procedure of de-ironing and re-ironing was tested for its possible 

 effect on the porphyrin or haemin. Porphyrin a of ratio bands III/IV 2-31 

 was converted to the haemin and again de-ironed. The ratio of the final 

 porphyrin a produced was 2-24 and the band positions had not changed. 

 This indicates that de-ironing and re-ironing by these methods had a negligible 

 effect on the compound. 



Paper Chromatography of Porphyrin a 



The porphyrin a purified by the method outlined here and after esterifica- 

 tion gave only one spot in ascending paper chromatograms using the solvent 

 systems, kerosene-propanol or light petroleum-acetone. However, previous 

 to the heat treatment step in the purification two spots of about equal intensity 

 were invariably obtained. When, after heating, the porphyrin was chromato- 

 graphed on a cellulose column it could no longer be eluted with ether but 

 required acetone. The porphyrin a in this eluate, esterified and run on paper, 

 gave only one spot corresponding to the slower of the two spots mentioned 

 above. After extraction into 17% HCI, however, the single spot obtained 

 corresponds to the faster of these two spots and the specific extinction of the 

 porphyrin is doubled. 



These experiments show that heat treatment alters the chromatographic 

 properties of the porphyrin-impurity complex and permits the separation of 

 porphyrin from impurity by extraction into 17 % HCl. They also demonstrate 

 that chromatographic studies of impure porphyrins may be misleading. 



EVIDENCE FOR A SINGLE PROSTHETIC GROUP FOR 

 MAMMALIAN CYTOCHROMES a AND a^ 



From the haemins extracted from ox heart we have obtained the following 

 porphyrins: protoporphyrin, cryptoporphyrin a and porphyrin ax. The 

 position of the absorption bands of cytochromes a and % rule out protohaem 

 as a possible prosthetic group for either cytochrome. We have been unable 

 to find any evidence indicating that porphyrin aa is a mixture of porphyrins 

 although differences in side chains not conjugated with the porphyrin ring 

 and therefore not affecting the spectrum are possible. 



The work of Parker (1959; see accompanying paper) and the demonstra- 

 tion by Morrison, Connelly and Stotz (1958) that cryptoporphyrin a exists in 

 acetone-HCl extracts of heart muscle as the haemin, strongly suggest that 

 cryptohaemin a is not an artifact produced during purification. However, a 

 large number of analyses in this laboratory indicate that the amount of 



