100 



J. H. Wano 



The films with more than 90 % polystyrene-content oxygenate and deoxy- 

 genate more slowly. The oxygenation property of these high polystyrene- 

 content films can be destroyed by heating at 70-80°C for an hour in an inert 

 atmosphere. This 'denatured' film can be reactivated by heating in carbon 

 monoxide atmosphere at 70-80°C for several hours, cooling to room tempera- 

 ture, and then removing the bound carbon monoxide by prolonged flushing 

 with nitrogen or by evacuation. The structures of the active centres in a high 

 polystyrene-content film at various stages of this process are depicted in Fig. 2 



5800 



5200 



Wavelength, A 



Fig. 1 . Spectra of a low polystyrene-content film with embedded haem groups. 

 Curve I, initial film, flushed with nitrogen gas for 3 hr 20 min after it was made to 

 remove the bound carbon monoxide; Curve II, the film was then flushed with 

 oxygen gas for 10 min; Curve III, the oxygenated fiJm was then flushed 2 hr 

 10 min with nitrogen gas. 



together with their absorption spectra. It is suggested in Fig. 2 that after the 

 film is activated by removing the bound carbon monoxide, the second imida- 

 zole group at each active centre is only weakly bound to the Fe''"+-ion because 

 of the constraint due to the solid-like matrix. Consequently this second imida- 

 zole group can be readily replaced even by a weak ligand such as the oxygen 

 molecule on exposure to air. By heating in an inert atmosphere, the solid-like 

 matrix softens. This allows the second imidazole group to diffuse, within a 

 short time, to the right position and orientation to form a stable co-ordination 

 bond to the Fe++-ion with maximum overlap of the atomic orbitals. Conse- 

 quently this 'denatured' film shows a sharper haemochrome-type of spectrum, 

 and is unable to combine with molecular oxygen. 



These results strongly support the hypothesis that the binding sites in 

 haemoglobin are largely covered with hydrophobic groups of the protein, 

 that each Fe++-ion is strongly bound to, presumably, an imidazole group on 

 one side of the haem plane, but is only loosely attached to another ligand on 

 the other side of the haem. This hypothesis is also consistent with the observed 



