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



(band II, asymmetric), 559 m/t (band III) and 518 m/< (band IV). The 

 ratio of bands III/IV in the best preparations is 2-40-2-45. The specific 

 extinction (optical density in 1 cm cell/mg porphyrin per ml; solvent ether) 

 is 20-24. From the molar concentration of the porphyrin, determined by 

 using the copper titration method of Oliver and Rawlinson (1951) and the 

 molecular weight derived from the values found by Warburg, Gewitz and 

 Volker (1955) for the haemin, pure porphyrin a was calculated to have a 

 specific extinction in ether at 559 mp, of 27. Our preparations thus contain 

 about 12-20% impurities. The millimolar extinctions found for porphyrin a 

 in ether at 559 m/t and 517 m/t are 21- 1 and 8-75 respectively. 



Attempts to crystallize the porphyrin using the above purification procedure 

 failed. Conversion to the haemin, which was held in ethereal solution in 

 concentrations up to 30 mg/ml at — 1 5° for about two months produced several 

 rosettes, clearly crystaUine, representing a very small proportion of the 

 total haemin. 



Purification as the Haemin 



Apart from the Warburg type preparation we have investigated methods 

 for preparing haem a from relatively small (2-4 kg) amounts of ox heart 

 mince. Purification procedures giving some success were: (1) partial precipi- 

 tation of phospholipids, (2) counter-current distribution between fight 

 petroleum-acetone and acetone-aqueous HCl (Kiese and Kurz, 1954) and 

 (3) chromatography on cellulose columns. It was clear from this work that 

 lipids are much more difficult to separate from haemin a than from porphyrin 

 a; also that the presence of lipid prevented complete separation of proto- 

 haemin and haemin a. The spectrum of the haemin a prepared by this 

 method was very similar to that prepared from our highly purified porphyrin a 

 except for some absorption due to contaminating protohaemin. 



Removal of Iron from Haemins {De-ironing) 



Careful examination of this procedure was necessary because it was found 

 that this step can be responsible for alteration of porphyrin a. 



The iron atom is replaced by two protons when the iron is ferrous and when 

 the proton concentration is high. The various factors affecting the rate of 

 this reaction have been described by Morell and Stewart (1956). Since their 

 publication it has been found that the formyl group of porphyrin a and other 

 formyl porphyrins can be oxidized to carboxyl by an unidentified oxidant 

 present in some batches of the acetic acid used as solvent for the haemin. A 

 high proton concentration accelerates the oxidation reaction which is 

 catalysed by ferrous, but not ferric, ions. The alteration of porphyrin a is 

 prevented by refluxing the acetic acid for an hour in the presence of ferrous 

 ions before distillation. 



