On the Stability of Oxyhaemoglobin 1 03 



Wang : Dehydration has little eflfect on the spectroscopic and oxygenating properties of 

 our films. But this could be due to the fact that our films are so hydrophobic that 

 they contain little water even before dehydration. Haurowitz's result is suggestive 

 that the sixth ligand in haemoglobin is water. He may be right, but I do not consider 

 his arguments as conclusive, since the conceivable structural changes in haemoglobin 

 caused by dehydration may release an imidazole group which has hitherto been 

 unavailable for close co-ordination because of constraints in the protein structure. 



(2) I hope that our denaturation experiments show some interesting resemblance 

 to the denaturation of haemoglobin. But there are also important inherent differences 

 between our film and haemoglobin. I hope that you will not carry the rather 

 interesting analogy too far. 



Legge: An old observation made by Robin Hill may be worth following up in relation 

 to the molecular environment around the haem or haemoglobin. The addition of 

 oxyhaemoglobin to chilled, concentrated KOH leads to the formation of a haemo- 

 chrome which, however, reverts to the native ferrous pigment when cautiously 

 neutralized with NH4CI. It may perhaps be easier to measure rates of autoxidation 

 and equilibria with ligands in such a system than in the analogous system (Zeyneck, 

 Haurov\ itz) where haemochrome formation is produced by removing water from dried 

 haemoglobin. Hill's system is at any rate a 'model' which appears to revert to the 

 original. 



George: There is am^ple kinetic evidence showing that the oxidation of ferrohaemoglobin 

 and ferromyoglobin to the ferric state by molecular oxygen are very complicated 

 chemical reactions. Although a free radical mechanism with single-equivalent steps 

 can be set up that leads to the observed rate equation it is more likely that two- 

 equivalent steps are involved (George, J. chem. Soc. 5436, 1954). 



An important feature, however, that undoubtedly contributes to the stability of 

 oxyhaemoglobin and oxymyoglobin toward electron-transfer fission is the exother- 

 micity of the formation of the oxygen complexes. Estimations of the ionization 

 potential of ferrohaemoglobin and ferromyoglobin in aqueous solution (Hanania, 

 Ph.D. Thesis, Cambridge, 1953) indicate that the reaction 



Fe++ + 02^ Fe+++ + O^ -f etc. (I) 



is endothermic: as a consequence, activation energy equal to or greater than this 

 endothermicity must be supplied for the reaction to occur. Now from a comparison 

 of reaction (1) with the electron transfer fission of the complex as in reaction (2) 



Fe++02 -^ Fe+++ -|- O^- + etc. (2) 



it can be seen that if Fe++02 is formed in an exothermic reaction from Fe++ and O2, 

 then the endothermicity of reaction (2) will be this much greater than that of reaction 

 (1). Since the exothermicity is at least 10 kcal/mole, this would make a very significant 

 contribution to the stability toward oxidation of ferrohaemoglobin and ferromyo- 

 globin relative to other ferrohaemoproteins and haem derivatives that might otherwise 

 have similar ionization potentials but not be capable of forming oxygen complexes 

 in exothermic reactions (George and Stratmann, Biochem. J. 51, 418, 1952). 



Lemberg: Myoglobin is more autoxidizable, more accessible to cyanide in its ferrous 

 form, and more ready to form mixed haemochromes with nitrogenous ligands than 

 haemoglobin. Its lyophobic matrix would therefore appear to be less dense. 



Wang: The nature of bonding between the oxygen molecule and haemoglobin in oxy- 

 haemoglobin has been a subject of controversy for many years. The structures 

 suggested by Pauling and by Griffith respectively are represented by A and B below. 



O 'O I r.OT-O- 



A B 



