174 J. E. O'Hagan 



Dodson, 1939). Spectrophotometric titrations of the combination of imida- 

 zole (Russell and Pauling, 1939) or hydroxyl (George and Hanania, 1953) 

 with ferrihaemoglobin also show linear relationships. None of the equations 

 proposed, on the basis of haem-haem interaction, to explain the sigmoid 

 oxygen dissociation curves, has been found to be satisfactory, except in a 

 special case at pH 9-1, outside the range of the Bohr effect (Roughton, Otis 

 and Lyster, 1955). 



There appears to be reliable evidence that specimens of mammalian haemo- 

 globins have at times exhibited hyperbolic dissociation curves (Barcroft, 

 1928; Hartridge and Roughton, 1925). Takashima (1955) found that at ionic 

 strength 0-03-0-3 the «-value for the Hill equation was about 3, which would 

 not be in accordance with Pauhng's equation. At lower ionic strength, pro- 

 nounced deviation from the Hill equation occurred, especially at the lower 

 portion of the curves. Rossi-Fanelli, Antonini and Caputo (1959) found that 

 human haemoglobin in 2 M sodium or potassium chloride (under which 

 conditions it is dissociated into half molecules), gave an oxygen dissociation 

 curve with slightly increased 'haem-haem interaction'. They did not show that 

 the four haems were divided between the two fragments but if they were, as 

 is most probably the case, their results could mean that the sigmoid curves of 

 haemoglobins were not due to interaction between the haems. 



Gastrophilus haemoglobin with two haems per molecule has a hyperbolic 

 oxygen dissociation curve, i.e. no 'haem-haem interaction' (KeiUn and 

 Wang, 1946). The sigmoid dissociation curve of diluted chlorocruorin (Fox, 

 1932) of molecular weight of about 3,000,000, with the possibility of inter- 

 action between about 200 haems (Lemberg and Legge, 1949), and the 

 'atypical' curves of haemoglobins of species such as the duck and carp (Red- 

 field, 1933), are difficult to reconcile with this hypothesis and have generally 

 been conveniently ignored. 



Some experimental support for the intermediate compound hypothesis 

 would appear to have been found from the work of Itano and Robinson 

 (1956) who detected intermediates when normal human adult carboxy- 

 haemoglobin was partly oxidized with ferricyanide and the mixture submitted 

 to electrophoretic separation. It does not necessarily follow, however, that 

 their findings are appUcable to the ferrohaemoglobin-oxyhaemoglobin 

 system. 



The finding by Hill and Holden (1926), Holden (1941) and Granick (1949) 

 of attachment of porphyrins to apohaemoglobins, the instability of aetio- 

 haemoglobin (O'Hagan, 1950, 1955, 1960) and the X-ray studies of ferri- 

 myoglobin (Kendrew et al., 1958) suggested the likehhood of linkages between 

 the haematin propionate side-chains and basic side-chains of the apohaemo- 

 globins and apomyoglobins. While studying the nature of these linkages it 

 appeared that it might be possible to explain more satisfactorily the oxygen 

 dissociation curves, the Bohr effect, the alkali-stabihty, and the differences 



