1. The action of a number of acids on four properties of gelatin (membrane potentials, osmotic pressure, swelling, and viscosity) was studied. The acids used can be divided into three groups; first, monobasic acids (HCl, HBr, HI, HNO3, acetic, propionic, and lactic acids); second, strong dibasic acids (H2SO4 and sulfosalicylic acid) which dissociate as dibasic acids in the range of pH between 4.7 and 2.5; and third, weak dibasic and tribasic acids (succinic, tartaric, citric) which dissociate as monobasic acids at pH 3.0 or below and dissociate increasingly as dibasic acids, according to their strength, with pH increasing above 3.0.
2. If the influence of these acids on the four above mentioned properties of gelatin is plotted as ordinates over the pH of the gelatin solution or gelatin gel as abscissæ, it is found that all the acids have the same effect where the anion is monovalent; this is true for the seven monobasic acids at all pH and for the weak dibasic and tribasic acids at pH below 3.0. The two strong dibasic acids (the anion of which is divalent in the whole range of pH of these experiments) have a much smaller effect than the acids with monovalent anion. The weak dibasic and tribasic acids act, at pH above 3.0, like acids the anion of which is chiefly monovalent but which contain also divalent anions increasing with pH and with the strength of the acid.
3. These experiments prove that only the valency but not the other properties of the anion of an acid influences the four properties of gelatin mentioned, thus absolutely contradicting the Hofmeister anion series in this case which were due to the failure of the earlier experimenters to measure properly the pH of their protein solutions or gels and to compare the effects of acids at the same pH of the protein solution or protein gel after equilibrium was established.
4. It is shown that the validity of the valency rule and the non-validity of the Hofmeister anion series for the four properties of proteins mentioned are consequences of the fact that the influence of acids on the membrane potentials, osmotic pressure, swelling, and viscosity of gelatin is due to the Donnan equilibrium between protein solutions or gels and the surrounding aqueous solution. This equilibrium depends only on the valency but not on any other property of the anion of an acid.
5. That the valency rule is determined by the Donnan equilibrium is strikingly illustrated by the ratio of the membrane potentials for divalent and monovalent anions of acids. Loeb has shown that the Donnan equilibrium demands that this ratio should be 0.66 and the actual measurements agree with this postulate of the theory within the limits of accuracy of the measurements.
6. The valency rule can be expected to hold for only such properties of proteins as depend upon the Donnan equilibrium. Properties of proteins not depending on the Donnan equilibrium may be affected not only by the valency but also by the chemical nature of the anion of an acid.