706 PHYSICAL CHEMISTRY OF THE PROTEINS. I 



For all these reasons we have come to consider the solubility of a 

 protein at its isoelectric point as a fundamental physicochemical 

 constant, characterizing and identifying the protein under investiga- 

 tion. We will attempt in subsequent communications to relate this 

 constant to the amphoteric dissociation of the proteins. 



'experimental. 

 The Purification of Protein. 



The three proteins whose isoelectric solubilities have thus far been 

 determined, serum globulin, tuberin, and casein, were each purified 

 as far as possible from other proteins, from multivalent anions and 

 cations, and from all but the last trace of electrolytes, by special 

 methods adapted to the nature of each. They were first prepared either 

 as ammonium or as sodium compounds. Analyses were then made 

 of the amount of ammonia or of sodium in these compounds, and the 

 amount of hydrochloric acid required to neutralize the base and pre- 

 cipitate the protein at its isoelectric point^" was calculated. 



The acid used was usually 0.01 n hydrochloric acid, and was de- 

 livered very slowly from a capillary tip extending well into the solution. 

 The solution was continuously and rapidly mixed by a motor-driven 

 glass screw-shaped stirrer which constantly forced fresh portions 

 of the protein past the glass tip from which the acid was being de- 

 livered, in a manner similar to that described by Baker and Van Slyke 

 (37). In this way the protein was never exposed to the denaturing 

 effect of a local excess of acid. After precipitation had begun, the 

 process of neutralization was further retarded in order to allow new 

 states of equilibrium to be fully attained. As the end was approached 

 small samples were removed, and the hydrogen ion concentration 

 electrometrically detennined. 



The isoelectric reaction was usually reached, at least in the case of serum globu- 

 lin, before the calculated amount of base had been neutralized. The base in 



The measurements that have been made thus far suggest that when the 

 hydrogen ion concentration of such a protein solution varied it moved in the 

 direction of neutrality. 



^^Loeb has recently discussed the significance of the isoelectric point for the 

 purification of protein (36). 



