H. GUTFREUND 



dissociation of both horse and human haemoglobin in dilute solutions 

 from ultracentrifugal observations. Only for foetal sheep haemoglobin 

 have results of osmotic pressure measurements on haemoglobin solu- 

 tions shown signs of dissociation on dilution 7 , and some preliminary 

 ultracentrifugal studies (Gutfreund, unpublished observations), showed 

 that molecules of this protein tend to dissociate into four sub-units. 

 There is certainly a marked difference between haemoglobins from 

 different sources in their tendency to dissociate on dilution ; with 

 adult sheep haemoglobin no dissociation effect could be detected on 

 ultracentrifugal examination of solutions containing 0-1 per cent 

 protein ; at this concentration a marked drop in the sedimentation 

 constant can be observed for horse and human haemoglobin. 



Solvents of different compositions have been used to study the 

 dissociation of haemoglobin ; among the most prominent of these are 

 aqueous solutions of urea and guanidine. The osmotic pressure of 

 protein solutions of finite concentration, however, does not only 

 depend upon the molecular weight of the protein ; this point will be 

 discussed in greater detail later in this paper. It is very difficult to 

 determine the haemoglobin concentration really accurately in the 

 presence of large quantities of urea or guanidine. Another medium 

 which is reported to cause dissociation of haemoglobin molecules in 

 solution, namely concentrated aqueous sodium chloride, was therefore 

 chosen for a preliminary investigation to differentiate between the 

 effect of protein dissociation and solvent-solute interaction on the 

 osmotic pressure of protein solutions. 



EXPERIMENTAL PROCEDURE 



The osmotic pressure of haemoglobin solutions was measured in the 

 simple osmometer described by G. S. Adair 2 . The protein concen- 

 tration, after equilibration, was in each case determined by the 

 micro-Kjeldahl procedure using all the precautions recommended by 

 A. C. Chtbnall, M. W. Rees and E. F. Williams 10 as well as their 

 value of 16-8 per cent as the nitrogen contents of haemoglobin. The 

 variations of the nitrogen contents of haemoglobins from various 

 species is within the experimental error of the methods employed. 

 The effect of large amounts of chloride on the nitrogen estimation was 

 checked by digesting ammonium sulphate with sulphuric acid and 

 catalyst in the same manner as the haemoglobin samples, and with 

 similar quantities of sodium chloride and lithium chloride. It was 

 found that the results were not affected by the addition of chlorides. 



The osmotic pressure was recorded in cms of water, calculated, 

 after correction for capillarity and density of the solution, from direct 



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