The Haem-Globin Linkage 1 77 



Attachment of Haematin and Haem to Human Serum Albumin 



Haematin combines with human serum albumin to form ferrihaemalbumin 

 (see discussion by Lemberg and Legge, 1949). J. Keilin (1944) considered 

 that the attachment of both haematin and haem to the albumin was through 

 the porphyrin and Lemberg and Legge indicated the haematin carboxyls as 

 being most hkely involved. O'Hagan (1955, 1960) in showing by both 

 spectrophotometric and paper electrophoretic studies that mesohaematin 

 (but not aetiohaematin) combined, demonstrated that the carboxyls were 

 responsible. Keilin (1944) believed it most likely that haem is attached in 

 similar fashion, on account of the spectral differences of ferrohaemalbumin 

 from free haem and from the haemochromes. 



The extent of the combination of human serum albumin with haematin 

 and haem was investigated by measuring the increment in absorbance in the 

 Soret region on addition of the pigments to excess of the albumin in a series 

 of buffer solutions of constant ionic strength. Tubes of 7 ml capacity were 

 filled with 5 ml buffer (phthalate or phosphate), 1 ml 2-5% human serum 

 albumin (the 25% solution diluted 1:10 with distilled water) and 0-1 ml 

 2x 10~^M freshly prepared protohaematin solutions (1-30 mg haemin 

 dissolved in 1 ml 005 n NaOH, then 9 ml distilled water added). Other sets 

 of tubes, one containing water in place of albumin and another with albumin 

 but no haematin were set up at the same time. The solutions were stood at 21° 

 for 3 hr, then portions transferred to a 10 mm cuvette and the absorbances 

 read at 404 m/f in the spectrophotometer. Another series of three sets of 

 tubes had 0- 1 ml freshly prepared 1 % w/v dithionite added to each tube and 

 the tubes closed by long stoppers which excluded air almost completely, 

 except for a small bubble which assisted mixing on inversion. After the 

 3 hr standing, portions of these solutions were carefully transferred with a 

 Pasteur pipette and as little agitation as possible, to the cuvette and the 

 absorbances read at 414 m/i, the Soret peak of ferrohaemalbumin. 



The curves obtained in Fig. 1 show the increment in absorbance due to the 

 addition of the albumin, i.e. they represent Aj^^ — A^ — A^, where ha = ferri- 

 or ferrohaemalbumin, h = haematin or haem and a = albumin. 



The difference in the attachment of the haematin and haem, as indicated 

 by the increment in absorbance, is at once apparent. Similar results were 

 obtained for the ferrohaemalbumin when the dithionite was added to the 

 ferrihaemalbumin after it had stood for 3 hr and the mixture stood a further 

 3 hr. No trace of verdohaem compounds was detected, but these were 

 formed, as expected, when the reduced solutions were reoxidized, so that the 

 reverse reaction (ferro- to ferrihaemalbumin) could not be investigated under 

 these conditions. To check whether the dithionite itself interfered in any 

 way, nickel mesoporphyrin (on which dithionite has no effect) was added to 

 the albumin and the absorbance increment measured before and after addition 



