170 



HARVEY A. IT.A.NO 



creasing amounts of a protein mixture were added to aliquots of a salt solution 

 of fixed composition at a given temperature and pressure. After equilibrium 

 was attained, the amount of protein in solution was measured and was plotted 

 against the total protein added. Characteristic curves were obtained for a 

 homogeneous protein, a two-phase protein mixture, and a solid solution of two 

 proteins. I have shown by use of this technique that the solubility behavior of 

 a mixture of hemoglobins A and S corresponds to that of a solid solution (Itano, 

 1953c). The solubility curve does not have any sharp breaks that would indi- 

 cate the appearance of a new phase, and the solubilities lie between those of A 

 and S. 50 mg of hemoglobin A dissolves completely in 10 ml of 2.24 molar 

 phosphate buffer. However, if a mixture containing 30 mg of hemoglobin and 

 20 mg of hemoglobin S is equilibrated with the same volume of buffer, the total 

 hemoglobin in solution is only 14 mg. This result indicates that a stabilizing 

 interaction in the solid phase prevents more than half of the available hemo- 

 globin A from going into solution. 



Kunitz and Northrop calculated the solubilities of solid solutions on the 

 assumption that the solubility of each component was proportional to its 

 solubility in its pure state multiplied by its mole fraction in the solid phase 

 and obtained fairly good agreement with theory when they tested two diflferent 

 chymotrypsin mixtures. I have carried out calculations based on the same as- 

 sumption for hemoglobin mixtures for which comparable data were available. 

 The results are summarized in Table II. A mixture of hemoglobins A and C 

 behaves according to the criterion of Kunitz and Northrop. Both the A-S and 

 S-C mixtures are less soluble than expected on the basis of the same criterion. 

 Moreover, although hemoglobin C is more soluble than A, the S-C mixture 



TABLE II 

 Calculated and observed solubilities of amorphous ferrohemoglobin 



1 Potassium phosphate buffer at 25° C was used. The mole fraction of K2HPO4 was 0.58 

 (Itano, 1953c). 



2 The calculations were based on the assumption that the solubility of each component is 

 equal to its solubility as a homogeneous protein multiplied by its mole fraction in the solid 

 state. (Kunitz and Northrop, 1938). Data for the calculations are from Itano (1951, 1953c). 



3 The observed solubilities shown here are means of several samples having the same 

 approximate compositions (Itano, 1953c). 



