Chinese Journal of Physiology

76 H. WU anv T. T. CHEN

albumins by means of tungstic acid would contain different amounts of ovomucoid and would give an erroneous picture of the amount of non-protein chromogenic substances liberated from the egg albumin. In the present study the egg albumin preparation used was ‘highly purified, and probably free from ovomucoid but to avoid the possibility of differential adsorption of this substance tungstic acid was not used for the preparation of the non-protein filtrates from the heated albumin solutions. .

Egg albumin was recrystallized six times and dialyzed in the presence of toluene in collodion tubes and chloroform in the water outside. An approximately 0.5 per cent solution was prepared and to 40 cc portions of this solution graded amounts of N/10 HCl or NaOH were added. Two sets of such solutions were prepared. One set was used for pH determinations and the other set was plunged into a bath of boiling water and allowed to remain there for 15 minutes. After cooling, the solutions were neutralized with sodium carbonate or acetic acid and proper amounts of water were added to equalize the volume (48 cc). The protein that was thus coagulated or denatured and flocculated was removed by filtration. The filtrates were heated once more in boiling water for 10 minutes.

The purpose of the second heating is to remove any trace of conalbumin which might be present in the albumin preparation. Conalbumin is not denatured by heating in slightly acid solution, but it can be coagulated by heat at its isoelectric point. It may be reasonably assumed that the amount of non-protein chromogenic substances liberated from the trace of conalbumin by this second heating is quantitatively negligible.

Any coagulum that might be formed on the second heating was removed by filtration. A non-protein filtrate from unheated albumin was prepared by adding to 40 cc of the albumin solution 2 cc H,O and 3 cc each of 10 per cent sodium tungstate. and 2/3 N sulphuric acid. For the determination of non-protein chromogenic substances, to 25 cc portions of the filtrates were added 0.5 cc of phenol reagent and 8 cc of 20 per cent Na,CO,. Tyrosine solutions were used as standards. The results are shown in table 1 and fig. 1.

If any ovomucoid were present in the albumin solution, it would be carried down by the tungstic acid precipitate in the control and not by the heat coagulum. The filtrate from the unheated control would contain less total chromogenic substances than that from the albumin