CHAPTER III 



METHODS OF DETERMINING THE ISOELECTRIC POINT 

 OF PROTEIN SOLUTIONS 



The results of the preceding chapter make it clear that when- 

 ever work with amphoteric electrolytes is contemplated it 

 becomes necessary to ascertain first the isoelectric point of the 

 substance, since at the isoelectric point the material can be most 

 easily freed from ionogenic impurities. There can be no doubt 

 that many of the substances exhibiting colloidal behavior are 

 amphoteric electrolytes. 



Hardy and Michaelis determined the isoelectric point by 

 observations on the migration of particles in the electrical field. 

 There are other methods available for this purpose, some of 

 which are often more convenient than Hardy's original method. 

 These methods are based on the fact that at the isoelectric point 

 the osmotic pressure, the viscosity, the amount of alcohol required 

 for precipitation, the conductivity, the swelling, the P.D. are all 

 a minimum. When the curves representing the values of these 

 properties are plotted as ordinates over the pH as abscissae, the 

 curves show a sharp drop at the isoelectric point. If, therefore, 

 a protein is brought to different pH by adding acid or alkali, and 

 if any of the properties mentioned is determined, the approximate 

 position of the isoelectric point can be inferred from the minimum 

 point of the property which is used as a test. The writer has 

 found it most convenient to use osmotic pressure experiments in 

 the case of proteins. 



The following older experiment by the writer may serve as an 

 illustration. 1 A number of doses each containing 1 gm. of finely 

 powdered Cooper's gelatin which had a pH of a little over 7.0 

 and consisted partly of Ca gelatinate were put for 30 minutes at 

 15C. into beakers containing 100 c.c. of HBr of different con- 

 centrations, varying from M/8 to M/8,192; and as a control 1 gm. 

 of gelatin was put for 30 minutes at 15C.into 100 c.c. of distilled 



1 LOEB, J., J. Gen. PhysioL, vol. 1, p. 363, 1918-19. 



37 



