548 CHEMICAL AND PHYSICAL BEHAVIOR OF CASEIN 



In our experiments we used casein prepared after Van Slyke and 

 Baker's method from skimmed milk and in addition from a com- 

 mercial "pure casein." Both preparations gave practically the same 

 result. In order to remove traces of fat from the casein the latter 

 was washed in acetone. 



2. In previous publications the writer had shown that weak dibasic 

 and tribasic acids combine in molecular proportions with crystalline 

 egg albumin, prepared after Sorensen and with gelatin.^ It can be 

 shown that the same is true for casein. 1 gm. of isoelectric casein, 

 prepared after Van Slyke and Baker, was put into 100 cc. of watery 

 solution containing 1, 2, 3, etc, cc. of 0.1 N HCl or 0.1 N H3PO4. The 

 pH of the casein solution was ascertained potentiometrically and the 

 number of cc. of 0.1 N acid required to bring the 1 per cent casein 

 solution to the same pH was plotted as ordinates over the final pH 

 of the casein solution as abscissae. The casein chloride or casein 

 phosphate is not completely soluble in a 1 per cent solution at room 

 temperature until the pH is about 3.0 or a trifle below. When too 

 much acid is added, i.e. when the pH is 1.6 or possibly a little above, 

 casein precipitates out again from a 1 per cent solution. 



Fig. 1 gives the curves for HCl and H3PO4, drawn out within those 

 limits of pH within which the casein salts are soluble in a 1 per cent 

 solution at room temperature. The curves show that about three 

 times as many cc. of 0.1 N H3PO4 as of 0.1 N HCl are required to 

 bring 1 gm. of originally isoelectric casein in a 1 per cent solution to 

 the same pH; or in other words, H3PO4 combines with casein in mole- 

 cular proportions, as we should expect if casein phosphate is a true 

 chemical compound. 



It was not possible to plot the corresponding curves for casein sulfate 

 and casein oxalate since these salts are too sparingly soluble. This 

 is true also for casein salts with other acids; e.g., triacetic acid. 



3. The writer had shown that the influence of different acids on the 

 physical properties of gelatin or crystalline egg albumin depends only 

 upon the valency and not upon the nature of the ion in combination 

 with the protein.^ Thus the values of osmotic pressure or viscosity 

 of gelatin chloride are identical with those of gelatin phosphate for 



3Loeb, J., /. Gen. Physiol., 1918-19, i, 559; 1920-21, iii, 85, 247; Science, 1920, 

 lii, 449. 



