184 J. E. O'Hagan 



position 6 to a greater or lesser extent than the one at position 7. Considera- 

 tion of the grouping together of rings 1 and 4 and 2 and 3 seems justified, and 

 there may be shared resonance between them as pairs, since on rupture the 

 ring sphts first at the a position to form verdohaems and the tetrapyrrohc 

 ring system of bihrubin sphts at the central methylene group on diazotization 

 (Lemberg and Legge, 1949). This could suggest that one propionate group 

 would be more affected by oxygenation than the other, so that if they were 

 joined by electrostatic hnkages to the apoprotein, one might be a labile link 

 under physiological conditions, while the other only split by such conditions 

 as high ionic strength, high urea or high hydrogen ion concentration, with 

 subsequent parting of the protein molecule into halves. 



Whatever the actual ipK values, those calculated by Altschul and Hogness 

 show that carbonic acid of pK = 6-352 at 25°C in water (Edsall and Wyman, 

 1958) could be displaced by a group or groups changing between minimum 

 p^ values of 6-5 and 5-8. The curves of Figs. 1 and 2 could suggest a change 

 of 0-9 unit, compared with a calculated change of 0-7 for two groups and of 

 1-4 for one group. 



The asymmetric haem could conceivably be attached to the apoproteins 

 directly or inverted so that structural isomers would be possible unless some 

 orientation by the side-chains occurred. Differences in the acid strength 

 of the two propionates might decide the orientation and also which group 

 could detach from the apoprotein on reduction. 



The curves obtained for the increment in the absorbance of nickel meso- 

 porphyrin on addition to apohaemoglobin show that a molecule of the size 

 and shape of haem can link by its propionate groups in the pH range 5-9 to a 

 residue in the apohaemoglobin not present in the carboxyhaemoglobin. A 

 specific structure in the proteins binding one or both of the propionates is 

 clearly indicated. That it is not the same as the one binding the iron atom can 

 be deduced from the work with aetiomyoglobin (Fig. 4). The measurements 

 given by Wyman (1948) of the apparent heat of dissociation of horse oxy- 

 haemoglobin and the work reported here, very strongly suggest that in this 

 species the groups binding the propionates are imidazolium side-chains. The 

 shape and range of the curves, and their similarity to the ones obtained for 

 combination with caffeine (Fig. 2), support this. 



Studies have not yet been extended to haemoglobins of other species; it 

 may be that other amino acid residues are responsible for bonding in these, 

 perhaps explaining the higher heat of dissociation given by Roughton (1944) 

 for ox haemoglobin at pH 6-8, and accounting for his findings in respect to 

 the carbamino reaction. 



The relationship between the new data, representing the attachment of the 

 propionate groups to the residue in horse apohaemoglobin, and the curve of 

 German and Wyman (1937) is shown in Fig. 5. The top curve of Fig. 5 was 

 obtained by subtracting the increments found for the attachment of nickel 



