708 



PHYSICAL CHEMISTRY OF THE PROTEINS. 



more than a few hours, and the precipitate allowed to settle. The 

 water-clear supernatant liquid was then decanted; distilled water 

 added approximately to the same volume; and the precipitate again 

 stirred for several hours. This operation was usually repeated from 

 six to fifteen times, and was only stopped when the wash water no 

 longer contained chloride, and when the amount of protein dissolving 

 in the wash water had become constant. 



The course of the purification of the euglobulin fraction of prepara- 

 tion rV, as measured by the protein nitrogen in an aliquot of successive 

 washings, is tabulated below. Approximately 14 gm. of this material 

 (14.72 gm. before neutralization) were triturated each day with dis- 

 tilled water. The total volume was 350 cc, and about 190 cc, or 

 slightly more than half of the clear supernatant liquid was decanted 

 each morning. It will be noted that at first the concentration of 



TABLE II. 



Purification of Serum Globulin IV h. 



soluble protein was also approximately halved each day, but that as 

 the purification proceeded, it approached a constant value. 



It is apparent that this method of purification is only applicable 

 to relatively insoluble proteins, since the loss of material would other- 

 wise be prohibitive. In the case of relatively insoluble proteins, how- 

 ever, it has the advantage of ensuring the identity of the product. 

 For unless two proteins have the same isoelectric point, and very nearly 

 the same solubility, this procedure may be used to effect their complete 

 separation. The more soluble protein dissolves in the successive 

 wash waters, and can be recovered from them by concentrating in 

 dialyzers, under negative pressure. It will, however, still contain 

 a trace of the less soluble protein. 



Recourse was first had to this method in the preparation of the 

 pseudoglobulin fraction of serum IV, When this fraction was 

 rendered isoelectric a large amount of protein was at first found in the 



