F. J. W. ROUGHTON 



where y is the percentage saturation 



p is the partial pressure of 2 (or CO) in mm Hg 

 K x , K 2 . . . are the equilibrium constants of the successive reac- 

 tions Hb 4 + X ^ HbA, Hb 4 Xi + X ^ Hb 4 X 2 etc. 

 where X = 2 or CO. 



The literature contains numerous examples, which will be summarized 

 later, of different sets of values of the unknown constants K u K 2 , K 3 

 and K 4 , all of which fit the observed data quite well provided that the 

 values of y are allowed a fairly generous margin of experimental error 

 (e.g. ± 3 per cent). For a more rigorous physico-chemical test of the 

 theory, the following conditions should be met : 



1 The haemoglobin concentration should not exceed 4 gm/100 cm 3 , 

 otherwise its osmotic pressure will not be proportional to the 

 concentration, and the law of mass action will therefore be in- 

 applicable without elaborate corrections. 



2 The estimates of y should be accurate to ± 1-5 per cent saturation 

 over the whole of the curve, and especially at the top and bottom, 

 since these regions turn out to be of critical importance. Sufficient 

 points must therefore be available in both these regions. 



3 The CO 2 content of the solution should be minimal, otherwise 

 there will be complications from the presence of carbamino com- 

 pounds which are thought to affect the O a -Hb equilibrium 1 . 



4 To reduce the chances of inactivation of the haemoglobin, the 

 temperature should not exceed 20°C, and the pH should not be 

 below 7-0. Actually a pW of ca 90 is very favourable, since in this 

 range the haemoglobin is still quite stable, and the dissociation 

 curve is not sensitive to slight variations in pH, as is the case at 

 neutral pH. 



5 The species of haemoglobin used should if possible be homogeneous 

 and not split into sub-units on dilution. 



Many of these conditions are far from ' physiological ', and since 

 much of the work on the oxy haemoglobin dissociation curve has been 

 done with physiological applications in mind, it is not surprising that 

 it fails to meet most of the requirements just listed, especially 1, 2 and 3. 

 The data of W. H. Forbes and F. J. W. Roughton (both published 

 and unpublished, 1931) comply, however, satisfactorily with conditions 

 1 to 4, and in part with 5. Their experiments were mostly done on 

 dilute solutions of adult sheep haemoglobin (0-3 to 0-5 gm/100 cm 3 ) in 

 borate buffer, pW 9-3, temperature 15-20°C. Two quite independent, 

 but concordant, methods of gasometric analysis were used. Figure 1 

 summarizes their best data, all reduced to the same scale of abscissae, 

 with p0 2 — 3 mm Hg at 50 per cent saturation (i.e. if in the case of a 



84 



