The Isolation, Purification and Properties of Haemin a 321 



acetone: 20% water (v/v), 50% acetone: 50% ether and 80% acetone: 20% 

 water. The haemins are extracted (once for 15 min, twice for 30 min) with 

 fresh lots of acetone-HCl and the haemins driven into ether by washing out 

 the acetone with dilute HCl. Undue contact of formyl-haemins with acetone- 

 HCl must be avoided since we have found that, in the presence of HCl, 

 acetone condenses with formyl groups. However, under the conditions 

 employed during this extraction the amount of acetone condensate should be 

 neghgible since experiment has shov/n that at room temperature and under 

 these conditions the reaction is complete only after 60 hr. 



Evaporation of the ethereal solution of haemins to about 1500 ml precipi- 

 tates much of the remaining protohaemin without appreciable loss of haemin 

 a. Most of the cryptohaemin a is also precipitated at this step. The phos- 

 pholipid content of the haemin solution is very greatly decreased by filtration 

 from solutions of the haemin in small volumes first of acetone-ether and then 

 acetone, both chilled at — 15°C. 



The haemins are converted to the porphyrins by the ferrous sulphate 

 method of Morell and Stewart (1956). Protoporphyrin and cryptoporphyrin 

 a are separated from the porphyrin a by extraction into aqueous 7 % (w/v) 

 HCl solution. The ethereal solution now contains the porphyrin a together 

 with some unsplit protohaemin. These are applied in solution in 50% 

 ether-50% light petroleum to cellulose columns which are developed with 

 ether. Much yellow lipid runs through the column near the solvent front, 

 followed by traces of protoporphyrin if still present and by porphyrin a. 

 Protohaemin remains on the column. The chromatography of this porphyrin 

 a is repeated; fractions of ratio of band III/IV 2-30-2-35 are combined for 

 the next step. 



Ethereal solutions containing about 20 mg of porphyrin a are evaporated 

 to dryness, spreading the porphyrin as thinly as possible on the walls of the 

 flask. The flask is then heated to 60°C under vacuum for 30 min. This treat- 

 ment permits the porphyrin a to be separated from much of the remaining 

 lipid by extracting the porphyrin from ethereal solution into 17% HCl 

 (w/v). This extraction is carried out in a cold room near 0°C to minimize the 

 formation of porphyrin a(i (see below). Porphyrin a(i is formed from 

 porphyrin a by standing at room temperature in strong aqueous HCl solution, 

 it is extracted from ether by 7% (w/v) aqueous HCl whereas porphyrin a 

 requires about 15% HCl. Both forms have similar spectra and the chemical 

 change is as yet unexplained. Porphyrin a^ is converted to porphyrin a 

 (porphyrin ay.) by increasing the temperature to about 60°C but some 

 porphyrin is destroyed (Lemberg, 1955; Lemberg and Stewart, 1955). 



The porphyrin a solution in 17% aqueous HCl is washed with ether and 

 the porphyrin returned to ether by adjusting the aqueous HCl solution to 8 % 

 followed by vigorous shaking. This porphyrin a solution now has bands at 

 647 mil (band I — barely discernible in the hand spectroscope), 578 m/t 



