INDICATOR METHOD 81 



probably combined in some proportion with the protein, which 

 may thus serve in a double manner to protect them from de- 

 struction. This fact, however, only adds to the complexity of 

 the conditions, without militating against the correctness of the 

 view urged by the investigators quoted above that the protein 

 protects the enzyme by binding some of the excess of acid or base. 



9. The Indicator Method. — This is simply the ordinary 

 method of acidimetry or alkalimetry applied to solutions contain- 

 ing protein, and has been very extensively used; unfortunately, 

 in the past, without a very clear understanding of the exact sig- 

 nificance of the data obtained. In order to appreciate this fact 

 it is necessary to recollect that the method, as ordinarily applied, 

 is not a static one. Not only is a foreign substance, namely 

 the indicator, added to the system under investigation, but while 

 acid or alkali is being added to the system to secure neutrality 

 to the chosen indicator, the combining capacity of the protein 

 is continually changing in response to the altering reaction of its 

 solution. The final result obtained in this manner with a given 

 indicator tells us nothing save the condition of equilibrium in 

 the solution at the precise H+ or OH' concentration at which that 

 indicator changes color; it cannot yield us information concern- 

 ing the acid or alkali binding capacity of the protein at any other 

 H+ or OH' concentration. In the further application of this 

 method, the methods of acidimetry and alkalimetry devised by 

 Friedenthal and Salm (14) (13) (44) and elaborated by Sorensen 

 (48) are indubitably destined to prove of the greatest utility, and 

 arouse the hope that the indicator method may be of more service 

 to the protein chemist in the future than it has been in the past. 



The use of indicators in protein solutions is, however, accom- 

 panied by two notable drawbacks. In the first place, owing to 

 the amphoteric character of the proteins and also to their mul- 

 tiple combining capacities, the changes in the hydrogen- or 

 hydroxyl-ion content of protein solutions, upon the addition of 

 acid or of alkali, over a considerable range, are relatively slight, 

 and, for this reason, sharp end-reactions are rarely obtained with 

 indicators in protein solutions, unless their color changes occur at 

 H+ or OH' concentrations lying without or upon the boundaries of 

 this range. Then, again, many of the substances commonly used 

 as indicators in acidimetry and alkalimetry combine chemically 

 with the proteins, and the compounds thus formed are not in- 



