246 VI. HEMOGLOBIN 



for the molecular weight. The value of 68,000 found for a salt-free 

 4% solution of hemoglobin agrees excellently with a molecule com- 

 prised of four subunits of weight 16,700. By working at low temper- 

 ature, Adair was able to avoid bacterial contamination during the 

 period necessary for the system to reach equilibrium; control of pH, 

 careful purification of the protein, and recognition of the Donnan 

 equilibrium enabled him to avoid the irregularities disturbing the 

 results of the early workers. Adair's value for the molecular weight 

 of mammalian hemoglobin was soon afterwards confirmed by Sved- 

 berg {2716,2720) with the ultracentrifuge. Other physical methods 

 such as surface tension {ISIfS), diffusion ( 1637, 20 J^5, 2058, 2809), and 

 ultrafiltration (659) have subsequently given results for the size and 

 molecular weight of hemoglobin which, within certain limits of pH 

 and concentration, agree well with those obtained by the ultra- 

 centrifuge or by the measurement of osmotic pressure. 



In a salt-free medium, Adair found the osmotic pressure to increase 

 on either side of the isoelectric point, pH 6.8, but observed that in 

 the presence of salt concentrations greater than 0.01 M the osmotic 

 pressure remained unaltered between pH 5 and pH 11. The osmotic 

 pressure is independent of the protein concentration up to 4%, but 

 beyond this it steadily rises (7), probably due to the departure of 

 the system from an ideal solution (382). 



4.1.2. Dissociation of the Hemoglobin Molecule. When they 

 reported on the molecular weight of hemoglobin, Svedberg and 

 Nichols did not investigate the molecular weight of the dissociation 

 products of hemoglobin found outside the pH stability zone, which 

 they found to lie between pH 6.0 and pH 9.8, a somewhat narrower 

 zone than that found by Adair. 



In 1930 Burk and Greenberg (382) examined the osmotic pressure 

 of purified horse hemoglobin in 6.66 M urea. Between pK 7.3 and 9, 

 the molecular weight proved to be 34,300, half that found in the 

 absence of urea. Below pH 7.3 the osmotic pressure of hemoglobin 

 increased in a manner similar to the behavior of salt-free hemoglobin 

 when the pH falls below 6.7. The protein was found to have the 

 molecular weight of 68,000 when measured in strong glycerol solution. 

 Wu and Yang (3131) confirmed Burk and Greenberg's results for 

 horse hemoglobin, but found that the hemoglobin of sheep and dog 

 did not dissociate in urea. The effect of urea and other amides has 

 more recently been investigated by Steinhardt (2621), who deter- 



