12 THE GENERAL CHARACTERS OF THE PROTEINS 



three further fractions were obtained, viz. : (i) " A " fraction, which is 

 completely thrown out by 62 per cent, of complete saturation, and 

 which is characterised by the relatively large amount of sulphur ; (ii) 

 " B " fraction, which is characterised by the intensity with which it 

 gives the furfurol or Molisch-Udransky reaction (see p. 32), and which 

 is precipitated by complete saturation in neutral solution ; and (iii) 

 " C " fraction, which is precipitated from the filtrate from " B " by 

 the addition of sulphuric acid solution saturated with ammonium 

 sulphate. By repeated solution and reprecipitation, and treatment of 

 the precipitates with alcohol, Pick succeeded in obtaining fractions 

 which differed from one another, not only in their solubility and pre- 

 cipitability, but also in their elementary chemical composition. 



Similar results have been obtained by Zunz with the use of zinc 

 sulphate. 



Another method of " salting out " has been suggested by Pinkus, 

 who recommends the saturation of the solutions with anhydrous 

 sodium sulphate at 37 C. 



The fractionation method, as employed by Pick and Zunz, has 

 recently been subjected to a critical examination by Haslam. 



It can hardly be contended that a process of the nature of frac- 

 tional precipitation can readily produce a complete separation of 

 fractions. Two sources of error are manifestly a priori conceivable, 

 viz. : (i) any fraction produced by a particular degree of saturation is 

 not absolutely insoluble in a solution of the strength in which it is 

 precipitated ; (ii) a precipitate may carry down with it certain quanti- 

 ties of substance which belong to a subsequent fraction. In a separa- 

 tion of globulin from albumin, for example, the precipitate produced 

 by half-saturation with ammonium sulphate will consist principally 

 of globulin, but it will contain certain quantities of albumin ; the fil- 

 trate, on the other hand, will contain mainly albumin, but it will not 

 be entirely free from globulin, which is not absolutely insoluble in 

 half-saturated ammonium sulphate solution. Two methods have been 

 suggested by Haslam for testing the purity of a fraction. 



I. Where it is sought to prove that a protein precipitate is free 

 from the substances of the filtrate. 



The precipitate is dissolved in water and the whole made up to 

 a given volume ; the amount of salt is added which is necessary to 

 produce the requisite degree of saturation ; the mixture after stand- 

 ing for twenty-four hours is filtered. If the filtrate contain no 

 protein, or other substance from which the precipitate is to be freed, 

 the requisite proof is furnished. If not, the organic nitrogen is 

 estimated by Kjehldahl's method. The precipitate is then re- 

 dissolved, the solution made up to the volume in which the original 

 precipitation was performed, the same amount of salt is added, and 

 the mixture is allowed to stand twenty-four hours, and finally filtered. 

 The organic nitrogen in the filtrate is estimated. This process of 

 precipitation at constant volume is repeated until the nitrogen in the 

 filtrate is a constant, i.e., until only that amount of protein is 

 present which is dissolved from the precipitate by the given volume 

 of the solution of the degree of salt saturation in which the precipita- 

 tion is carried out. This process of precipitation at constant volume 

 must generally be repeated several times before a homogeneous 

 precipitate is obtained. 



