354 R. Lemberg, P, Clezy and J. Barrett 



HAEM A2 AND CHLORIN A^ 



Haemin a., (Barrett and Lemberg, 1954; Barrett, 1956) has been isolated 

 from all micro-organisms in which the cytochrome a^ band at 630 m/i could 

 be observed, and in small concentration in a few in which it had previously 

 not been observed, e.g. Bacillus subtilis. Its amount paralleled the strength 

 of the cytochrome Ao band. Further, anaerobically cultured bacteria con- 

 taining no cytochrome a^ also failed to yield haemin a^- 



Aerobacter aerogenes was our main source. The bacterial haemins in acid 

 ethereal solution showed an absorption band at 630 m/f not found in the 

 spectrum of protohaemin. This was used as guide in the isolation procedure 

 as ^603m///'£^635m/i- This Varied from 1-3 in cells with much cytochrome a^ 

 to 1-0 in those with little, corresponding to a protohaem/haem a^ ratio of 

 from 4-10. 



The separation of the green haemin from protohaemin was achieved by 

 silica gel chromatography and freezing out of protohaemin together with 

 phospholipids at — 70°C from acetone solutions. Purified haemin a^ had an 

 ^603m;./^635m,. ratio of 3-18. 



Iron is removed more readily from haem a^ than from protohaem and even 

 from haem a. This process could therefore be carried out with relatively low 

 HCl concentration (0-4 % w/v) at room temperature. This yielded a chlorin 

 (dihydroporphyrin). It is known that metals are less strongly bound by 

 dihydroporphyrins than by porphyrins. Re-introduction of the iron was 

 correspondingly more difficult, but restored haemin a.^- 



Further purification was achieved by silica gel chromatography, counter- 

 current distribution in aqueous methanol-1 % HCl and fight petroleum, 

 using as a guide the ratio £'653m///-^503m// which is 3-3 in the purest chlorin, and 

 by HCl-fractionation. The last traces of lipid are held very fast, as by 

 porphyrin a, and haemin and chlorin a^ remained oily. It is therefore likely 

 that haemin ^3 contains a large alkyl side chain. Unfike haemin a, but fike 

 cryptohaemin a, it accompanies protohaemin in the Rawlinson separation. 

 Paper chromatography by the lutidine- water method of Nicholas and 

 Rimington (1949) shows that chlorin a^ is a dicarboxylic acid, but fike 

 porphyrin a its Rp (0-87) is somewhat higher than those of usual dicarboxyfic 

 porphyrins (0-80). 



Haem a^ does not contain a carbonyl side chain ; neither the haemochrome 

 nor the chlorin reacted with hydroxylamine. 



Like other chlorins, chlorin a.^ was converted into porphyrins by catalytic 

 hydrogenation and reoxidation, and by hydriodic acid. Neither of these 

 reactions proceeds, however, without alteration of side chains. Preliminary 

 experiments of dehydrogenation of vinylchlorins by photo-oxidation in the 

 presence of quinones have been carried out and this method is being studied 

 with chlorin ^2. The product of the catalytic hydrogenation closely resembled 



