THE RELATION OF PROTEINS TO CRYSTALLOIDS. II. THE OSMOTIC PRESSURE OF IONISING SALTS OF SERUM PROTEINS
Authors: Roaf H. E. 
Published: December 2014

1. Previous workers have shown that amino acids and proteins form salts with acids and alkalies which are ionised, and behave like the salts of fairly strong bases and acids. The presence of such salts must cause a high osmotic pressure, as the inorganic ions would be prevented from passing through a membrane impermeable to the protein, and at the same time an electrical potential would be established. As the salt hydrolysed the inorganic electrolytes formed would pass through the membrane, with consequent fall of osmotic pressure.

2. Measurements of freezing‐point and electrical conductivity of glycine with acids and alkalies fully confirm these conclusions. The degree of ionisation is not so great as that found with hæmoglobin, but it must be remembered that the so‐called uncombined electrolyte may include some ionised salt. Siegfried (12) points out that barium hydrate added to amino acids produces an alkaline solution from which barium cannot be precipitated by carbon dioxide. This points to a salt of the type BaR+ + OH−, and this with an equal amount of salt ionised after the type Ba+ + ROH− would correspond in the experimental measurements to equal quantities of unionised salt and uncombined alkali (i.e. due to hydrolysis). In that case the ionised salt would really have a higher value than that given in the protocols.

3. Osmotic pressure measurements show that the presence of excess of acid or alkali lowers the osmotic pressure of serum proteins. Removal of excess of inorganic electrolyte causes a rise of pressure. With alkali the logarithm of the maximum pressure is proportional to the logarithm of the amount of alkali present, thus corresponding to the curve shown by Adamson and Roaf (2). With acid the maximum pressure is practically the same for concentrations of acid from 0·016 N to 0·18 N, thus showing absence of hydrolytic cleavage of the protein.

4. Continued dialysis and removal of alkali by carbonic acid causes a more rapid fall of pressure than conditions that do not promote hydrolysis.

5. The explanation of these results is believed to be the formation of ionising salts of proteins with acids and bases. Excess of electrolyte diminishes ionisation and also causes aggregation of the colloid. Removal of excess of electrolyte promotes ionisation and diminishes the aggregation. When hydrolytic dissociation occurs the pressure falls as the inorganic ions pass through the membrane, and at the same time the protein may aggregate. Considered along with the results obtained with hæmoglobin, where the effect of aggregation can be controlled by its known molecular weight, the interpetration of the rise of pressure as mainly due to ionisation seems justified.

6. The formation of such salts in the body is possible. Normal tissues have a very low concentration of ions. Being practically neutral, they possess very few hydrogen or hydroxyl ions. This seems to be a universal rule for all ions, as potassium and chlorine ions are likewise absent from resting muscle and nerve. Physiological activity results in the setting free of ions of hydrogen potassium and chlorine in the case of muscle. If these ions are present as ionising salts with proteins, large osmotic pressures may be produced without postulating a membrane impermeable to inorganic ions. The small amount of ions that pass through the membrane before being held back by the opposite charge on the protein would account for the well‐known electrical potential which accompanies physiological activity. Owing to the duration of such processes as muscular contraction being very short, there would not be sufficient time for any appreciable loss of ions by hydrolysis.

7. These experiments differ from those with hæmoglobin because there was apparently a formation of hydrates so that the pressure per 1 per cent. was greater in the more concentrated solutions of protein. With hæmoglobin the pressure per 1 per cent. was greater in the more dilute solutions.

8. The evidence of an ionising salt of protein with sodium chloride is not decisive.

9. The pressures obtained with distilled water probably represent a condition of slight alkalinity, because at a certain point (near the true neutral point) the pressure is zero (1) and a precipitate occurs.