On the Stability of Oxy haemoglobin 99 



incorporated in poly-l-vinyl-4-pyrrolidone by copolymerization, are rapidly 

 oxidized on exposure to air. Thus the greater reduction potential of methaemo- 

 globin is, by itself, inadequate to explain the observed stability of oxyhaemo- 

 globin. 



The oxidation of haem by molecular oxygen may take place through one or 

 more reaction paths. If the initial step of the oxidation involves the formation 

 of 'oxyhaem' followed by the decomposition of the latter to ferrihaem and 

 Og" or HO2, one would expect the subsequent reduction of Og" or HO2 by 

 other haem molecules to be comparatively fast. Consideration of coulombic 

 interactions would predict the decomposition of 'oxyhaem' to ferrihaem and 

 Of or HO2 to be slow in non-acidic media of low dielectric constant. Thus 

 if one assumes that the binding sites in haemoglobin are not completely 

 exposed to water but are largely covered with hydrophobic groups of the 

 protein, one would expect the decomposition of oxy haemoglobin to methaemo- 

 globin and Og" or HO2 to be much slower than the corresponding process for 

 a freely exposed 'oxyhaem'. But one would expect the dissociation of such a 

 protected 'oxyhaem' back to haem and molecular oxygen to be practically 

 unhindered, although decomposition to products with net charge or high 

 polarity would be retarded by the comparatively non-polar nature of the 

 local environment. On the other hand, if the rate-determining step is a two- 

 electron oxidation involving two haem molecules, then these protective 

 hydrophobic structural elements could also effectively prevent the transfer of 

 electron between the two haem groups and hence retard the oxidation. 



RESULTS AND DISCUSSION 



In order to check the above hypothesis, synthetic models were made by 

 embedding the derivatives of haem in a lyophobic matrix (Wang, 1958). 

 The Fe++-ion in each haem group was bound firmly on one side of the haem 

 plane to a ligand molecule ; on the other side, it was bound only loosely to 

 another ligand molecule. These embedded haem groups combine reversibly 

 with molecular oxygen, stable even in the presence of water. 



These model materials were made by first preparing a solution of 

 l-(2-phenylethyl)-imidazole-carbonmonoxy-haem diethyl ester in benzene 

 containing dissolved polystyrene and an excess of l-(2-phenylethyl)-imidazole, 

 drying in a warm stream of carbon monoxide, and then removing the bound 

 carbon monoxide molecules by prolonged evacuation or flushing with an 

 inert gas at room temperature. The resulting bright-red, transparent film 

 showed a haemochrome-type of spectrum which varied somewhat with the 

 composition of the matrix. 



The spectra of a film made by imbedding such haem derivatives in a 

 matrix of 25 % polystyrene and 75 % l-(2-phenylethyl)-imidazole are shown in 

 Fig. 1. The oxygenation is, within experimental uncertainties, quantitatively 

 reversible. 



