2 20 Solubility of Proteins 



Effect of Other Proteins on Solubility 



The solubility of a given protein in a mixture of other proteins depends not 

 only upon the factors already considered, but also upon the presence and con- 

 centration of other proteins. These differences in solubility are difficult to 

 explain adequately but are undoubtedly due partially to the effect of the 

 other proteins on the electrostatic characteristics of the medium or possibly 

 to the combination of one protein with another. Whatever the reason, pro- 

 teins are usually more soluble in the presence of other proteins. Thus, pro- 

 teins completely precipitated in a given salt solution in the presence of other 

 proteins and other organic molecules found in an extract may require a 

 much lower concentration of salt for precipitation as purification proceeds. 



After a protein has been purified, the concentration of salt at which the 

 solubility will reach a given low value will be constant, but the point at which 

 precipitation first begins necessarily depends upon the concentration of the 

 protein. Precipitation starts when the concentration of the protein exceeds 

 the solubility of the protein under the given conditions, that is, when the 

 solution is saturated with respect to the protein. If pure proteins are precipi- 

 tated from a more concentrated protein solution than that found in the 

 original extract or mixture, beginning precipitation at a lower salt concen- 

 tration may be due to increased concentration of the protein itself rather 

 than to the removal of other proteins. 



One other consideration might be mentioned. Comparable proteins from 

 different species do not necessarily have the same solubility characteristics. 

 For instance, crystalline hemoglobin from human red cells, although having 

 the same molecular weight and the same oxygen-combining capacity as that 

 from the red cells of the horse, is one thousand times more soluble in phosphate 

 buffers of the same ionic strength and pH." Thus, it is usually not possible 

 to employ identical purification procedures in dealing with comparable 

 material from different species. 



Criteria for Purity 



Since the protein molecule is so large and since its actual chemical structure 

 is still a mystery, the tests for the identity and purity of a given protein fraction 

 are necessarily indirect. One of the most rigorous tests for homogeneity of a 

 given protein fraction is constant solubility under given conditions inde- 

 pendent of the amount of saturating body. Constant chemical composition 

 upon reprecipitation of proteins containing an inorganic constituent such 

 as iron, copper, sulfur, or phosphorus is also an excellent indication of purity. 

 One boundary in the electrophoresis cell and one molecular weight by diffu- 

 sion and sedimentation measurements, constant mobility, dielectric constant, 

 amphoteric properties, and viscosity all give additional information. Enzymes, 

 hormones, antibodies, and other proteins having physiological functions 

 which exhibit constant activity per milligram of protein upon repeated refrac- 



