268 Vr. HEMOGLOBIN 



the hemoglobin liy treatment with alumina cream. Under these conditions, 

 they found that the value of a was very sensitive to variations in ionic 

 strength. In salt-free preparations, a = 2, while, in bicarbonate buffer, 

 a = I'i. The dissociation constant. K. was similarly found to vary from 0.21 

 to 0.0057. They suggest that in salt-free solutions, the hemes might interact 

 only in pairs and were able to fit their data equally well on this assumption. 

 AVhile Altschul and Hogness were able to fit a given dissociation curve by 

 suitable choice of K and a, Roughton, in a personal communication, informs 

 us that the data of Forbes and Roughton (OlS) cannot be fitted by Pauling's 

 equation. In this work the dissociation curve was measured on solutions of 

 hemoglobin within the concentration range in which Adair's work had shown 

 the osmotic pressure to be proportional to concentration. Only in these 

 cases, as Adair has stressed, is it possible to use concentrations of hemoglobin, 

 rather than activities in applying the law of mass action; nevertheless this 

 point has been frequently ignored even in the theoretical discussion of the 

 data which have formed the basis for most of the recent work on the equi- 

 librium. 



Single values of K and a may be selected which enable the equation to fit 

 the top of the dissociation curve, or the bottom of the curve but which will 

 not fit the whole curve. Forbes and Roughton, indeed, were able to fit their 

 data by a modified Adair equation in which only Ki and K4 were determined 

 arbitrarily, while the values for K2 and K3 followed from tlmt chosen for Ki 

 according to statistical considerations deduced from a recasting of the Adair 

 equation in terms of Langmuir's adsorption theory. 



These workers point out that when p is small in the Adair equation, it 

 simplifies to: 



Kip 



^ ~ 4(1 + Kip) 



since p^, /»^ and p* can be neglected. When p is very large the equation 

 becomes : 



SKiKaKsp^ + 4X1X2X3X4/)^ 3 + 4K4P 



y 



4KiK2K3/r^ + 4X1X2X3X4^^ 4 + 4X4P 



and by accurate determination of the values at the upper and lower ranges 

 of the curve it might be possible to determine Ki and K; separately, leaving 

 only K2 and K3 to be determined arbitrarily. So far this has never been done. 

 A further problem is presented by the possibility that the dissociation 

 curve may be affected by as yet unknown factors. An example of this may 

 be found in the occasional reports of samples of mammalian hemoglobin 

 whose dissociation curve has been found to be hyperbolic. Such instances 

 are cited in Barcroft's monograph (141) and in papers by Hartridge and 

 Roughton {1H'>) and Forbes (OlS). These results have never been explained. 



No finality can therefore be .said to have been reached on the 

 choice of the most suitable equation to express the dissociation curve. 

 The reader must bear this qualification in mind M^hen we make use 



