THE ISOLATION, PURIFICATION AND PROPERTIES 



OF HAEMIN a 



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



Institute of Medical Research, Royal North Shore Hospital, Sydney 



This paper describes the isolation, purification and properties of the haemin 

 derived from mammahan cytochrome c oxidase. As will be shown, existing 

 evidence indicates that a single major haemin is obtained which is thus referred 

 to as haemin a. Evidence is discussed indicating that haemin a is also obtained 

 from cytochrome a^ of certain bacteria. 



PURIFICATION OF HAEMIN a AND PORPHYRIN a 



Warburg and co-workers (Warburg and Gewitz, 1951; Warburg, Gewitz 

 and Volker, 1955) purified haemin a from horse heart (without conversion 

 to the porphyrin at any stage) and obtained crystalline material. At the stage 

 before crystallization the yield was about 30%. Their method requires as 

 starting material 64 horse hearts from which about 140 kg washed mince are 

 obtained. 



Other workers, notably Rawlinson and Hale (1949), Lemberg and co- 

 workers (Lemberg, Falk, Rawlinson, Hale and Rimington, 1949; Lemberg 

 and Falk, 1951; Lemberg, 1953; Lemberg, Bloomfield, Caiger and Lock- 

 wood, 1955; Lemberg and Stewart, 1955), Granick (1952), Dannenberg 

 and Kiese (1952), have converted the haemin to the porphyrin during purifica- 

 tion but have not obtained significant amounts of crystalUne material. We 

 have preferred the porphyrin approach for a number of reasons. The scale 

 of the Warburg preparation is too large for our facihties, and attempts to 

 repeat this preparation on a smaller scale were not satisfactory and gave no 

 crystaUine haemin a. We prefer conversion of the haemin to porphyrin at an 

 early stage because alterations of the prosthetic group or the possible presence 

 of different prosthetic groups can be more easily detected in the characteristic 

 porphyrin spectra with their sharp and high absorption bands. 



Method 



We have found that about 18 kg ox heart mince (from about 27 kg ox 

 hearts) is a convenient amount of starting material. 



The mince is washed in water and saline to remove most of the haemoglobin 

 and myoglobin. It is then defatted by successive extractions with 80% 



320 



