JACQUES LOEB AND ROBERT F. LOEB 197 



precipitating effect of low concentrations of neutral salts? Let us 

 assume that the ultimate particles in a solution of casein chloride of 

 pH 2.2 are, (a) isolated casein ions, (b) isolated casein molecules, and 

 (c) small casein aggregates or micella. The Donnan equilibrium 

 furnishes two kinds of forces preventing that degree of coalescence 

 of these particles which is required for precipitation; namely, the 

 osmotic pressure and the membrane potentials. When isolated pro- 

 tein ions collide and remain attached to form a micellum, a Donnan 

 equihbrium is established between the nascent micellum and the 

 surrounding solution. The Donnan equilibrium demands that 

 there be a higher concentration of electrolytes inside than outside 

 and this difference in osmotic pressure leads to water being attracted 

 into the micellum. The increase in hydrostatic pressure will force 

 the protein molecules apart again and thus tends to prevent the forma- 

 tion of the micellum. Moreover, if micella exist in the casein chloride 

 solution (aside from isolated casein ions and molecules) the coalescence 

 of different micella into larger aggregates must be prevented by the 

 potential difference between the micella and the surrounding solution. 

 J. A. Wilson suggested in 1916^'' that the source of the charges might 

 be the Donnan equilibrium, and one of us has recently shown that 

 these potential differences between a gel and the surrounding solution, 

 which Donnan 's theory demands and which Wilson postulated, 

 actually exist. ^^ As a consequence of this p.d., the micella are charged 

 and must repel each other according to the charge. This charge 

 caused by the Donnan equihbrium is a minimum at the isoelectric 

 point, rises with increasing hydrogen ion concentration, reaching a 

 maximum, and diminishes again with a further increase in hydrogen 

 ion concentration as shown in a preceding paper. The osmotic 

 pressure and charge are also diminished by the addition of salt. 



Hardy's rule that only that ion of a neutral salt is active in 

 precipitation which has the opposite sign of charge as the colloidal 

 ion, and that the efficiency of the ion increases with the valency is 

 simply the expression of the Donnan effect, as is also the fact that 

 very low concentrations of electrolytes suffice for precipitation. The 

 reader will notice that it is unnecessary to assume that the ions 



>° Wilson, J. A., J. Am. Chem. Soc, 1916, xxxviii, 1982. 

 " Loeb, J., J. Gen. Physiol, 1920-21, iii, 557, 667. 



