14 THE GENERAL CHARACTERS OF THE PROTEINS 



Method of Fractional Precipitation. 



The limits of precipitability may be determined in the following 

 way. To 2 c.c. of a protein solution in a series of test-tubes are 

 added 8 c.c. of liquid containing varying quantities of distilled water 

 and saturated salt solution. The limits of incipient precipitation 

 can be readily observed. [Pick found in working with Witte's 

 peptone and ammonium sulphate solution that precipitation com- 

 menced when a mixture of 2*4 c.c. saturated ammonium sulphate 

 and 5 '6 c.c. water were added to 2 c.c. of a peptone solution. Such 

 a solution contains 24 per cent, of the salt necessary for complete 

 saturation of the whole 10 c.c. of liquid. In this case the initial 

 limit of precipitation may be expressed by the number 24.] The 

 contents of the other test-tubes containing a larger quantity of am- 

 monium sulphate are examined by filtering off the precipitate and 

 adding one or two drops of the salt solution to the filtrate ; as soon 

 as this addition no longer produces a precipitate, the higher limit of 

 precipitation is reached. In dealing with a mixture, a second pre- 

 cipitation often commences with the addition of larger quantities of 

 salts. This can be readily observed in the series of tubes which 

 contain a higher concentration, and the point is noted when the 

 addition of a drop of saturated salt solution to a filtrate produces a 

 precipitate again. This point is the lower precipitation limit of a 

 second fraction. The higher precipitation limit should also m this 

 case be noted. A third fraction can also be obtained and its precipita- 

 tion limits determined. [Pick showed that the first fraction, which 

 commenced to fall out when the liquid was 24 per cent, saturated (see 

 above), was. completely precipitated with 42 per cent, saturation, 

 i.e., the filtrate from the mixture 2 c.c. peptone, 4'2 c.c. ammonium 

 sulphate solution, and 3*8 c.c. water just failed to give a precipitate 

 when a drop of saturated salt solution was added ; when, however, 

 the 54 per cent, limit was reached, the filtrate just commenced to 

 give a precipitate with a drop of saturated salt solution. By the 

 method three fractions altogether were obtained, viz. y the 24-42 per 

 cent, fraction, the 54-62 per cent, fraction, and the 70-100 per cent, 

 fraction.] 



A pure protein gives reliable precipitation constants ; attention 

 must be called, however, to the criticisms of Haslam already outlined 

 above on the application of the salting-out process to the separation 

 of mixtures; as already noted, it is a tedious process to obtain a 

 fraction of constant composition by the method. 



Proteins, which are insoluble in water, but soluble in salt solu- 

 tions, have also their precipitation constants. Osborne, in his work 

 on the plant globulins, extracted his raw material with 10 per cent, 

 ammonium sulphate solution ; he then determined the precipitation 

 limits by increasing the concentration of this solution. The con- 

 stants were required to discover whether globulins derived from 

 different plants were identical or not. 



In determining the precipitation limits with animal liquids, such 

 as serum, it must be remembered that these already contain salts ; 

 in fact, the globulin can be separated from serum by dialysing these 

 salts away. 



