THE PHYSICAL PROPERTIES OF THE PROTEINS 13 



II. When the substance it is sought to purify is in the filtrate 

 (e.g., an albumin contaminated with globulin). 



In this case a process of fractional precipitation must be per- 

 formed. By " fractional precipitation " a different process is implied 

 to that suggested by Pick. The method employed by Haslam may 

 be best understood by giving a concrete example of its mode of 

 application, vis., the preparation of secondary or deutero-proteose. 

 The primary proteoses in Witte's peptone are first separated by half- 

 saturation with ammonium sulphate ; from the filtrate the secondary 

 proteoses are precipitated by adding ammonium sulphate to complete 

 saturation. This precipitate is then dissolved in water, and the 

 solution half-saturated with the sulphate; a smaller quantity of 

 primary proteose is precipitated than that obtained in the first pre- 

 cipitation ; from the filtrate the secondary proteoses are precipitated 

 again by complete saturation with the salt. These processes are 

 repeated until half-saturation with the sulphate no longer produces 

 a precipitate. Even now, the "secondary" proteose is not quite 

 free from the primary. To the half-saturated solution, which should 

 contain about 2 per cent, of proteoses, saturated salt solution is added 

 until a small precipitate appears. Practice will enable the operator 

 to judge how much substance it is best to precipitate at each " frac- 

 tionation". The fraction is then filtered off, dissolved in water so 

 as to make approximately a 2 per cent, solution, and to this is added 

 an equal volume of saturated salt (i.e., ammonium sulphate) solution. 

 A precipitate of primary proteoses (z>., the substance which it is 

 desired to separate off) will be produced ; this is filtered off, and the 

 filtrate is returned to the main solution. A second " fraction ' ' is then 

 taken from this by partial precipitation ; this fraction is dissolved in 

 water (to make 2 per cent, solution approximately), diluted with an 

 equal volume of ammonium sulphate (another precipitation of primary 

 proteoses) and the filtrate therefrom returned to the main solution. 

 This process is repeated, a small quantity of primary proteose being 

 removed each time from the solution, until a " fraction " no longer 

 gives a precipitate on half-saturation. The main solution is then 

 completely saturated with salt, and a precipitate thereby obtained 

 which consists of a nearly pure deutero-proteose. 



It will be seen from the above descriptions that the process of 

 obtaining by salt precipitation a protein of constant composition is 

 an extremely tedious one, and it is highly probable that most of 

 the proteins obtained by earlier investigators by the method of frac- 

 tional precipitation have been impure. 



It may be remarked here that the methods employed by Haslam 

 do not apply only to precipitation by salt solutions ; fractional pre- 

 cipitation by alcohol of different strengths may be carried out in a 

 quite analogous way. 



Little detail has been given above concerning the fractions obtained 

 by different observers from the products of digestion of proteins, such 

 as Witte's peptone ; this subject, it is to be hoped, will be treated more 

 fully in a monograph on digestion. Neither has anything been said 

 on the physical processes involved in the method of " salting out " ; 

 this, again, is foreign to the scope of this article, and should be treated 

 in the monograph which deals with the physics of colloidal solutions. 



