20 THE GENERAL CHARACTERS OF THE PROTEINS 



as before. On a second recrystallisation, needles were obtained 

 mixed with the spherolites ; on repeating the crystallisation a suffi- 

 cient number of times, a product consisting entirely of needles was 

 obtained. 



The original method of Hofmeister has been modified in various 

 ways. It has been shown that ammonium sulphate solution con- 

 taining protein becomes alkaline on standing ; Hopkins and Pinkus 

 have shown that the addition of acid facilitates very considerably 

 the process of crystallisation. The method as modified by these 

 investigators is the one now in general use and is carried out in the 

 following way : 



Egg-white is beaten to a froth (to break up the membranes) with 

 exactly its own bulk of ammonium sulphate solution. The mixture, 

 after standing overnight, or at least for a few hours, is filtered from 

 the precipitated protein. The filtrate is now measured. Ten per 

 cent, acetic acid (glacial acetic acid diluted to ten times its bulk) is 

 then very gradually added from a burette, until a well-marked 

 precipitate forms a precipitate sufficient to make the mixture 

 actually milky in appearance, and not a mere opalescence for which 

 liberated gas bubbles might be mistaken. The actual amount of 

 acid required to produce such a precipitate will vary (chiefly because 

 of the varying loss of ammonia which occurs when the liquid has 

 previously stood in open vessels). The point corresponds roughly 

 to an incipient acidity of the liquid towards litmus, but the formation 

 of the precipitate forms of itself the best indicator.^ This stage 

 being reached, a measured quantity of the acid is now added, over 

 and above that required to produce the first precipitate, I c.c. being 

 added for each 100 c.c. of the filtered mixture as originally 

 measured. The whole contains, therefore, approximately I part per 

 thousand of free acid. The bulky precipitate thus produced is at 

 first amorphous, and if the mixture be occasionally shaken the 

 amorphous precipitate will give place to crystals within four or five 

 hours. To obtain the full yield, however, the material should stand 

 for twenty-four hours. The product thus obtained is already nearly 

 pure. On recrystallising once more from ammonium sulphate 

 (dissolving in water, and then carefully adding half-saturated 

 ammonium sulphate containing acetic acid in the proportion of I 

 per thousand, till a permanent precipitate forms, and then about 

 2 c.c. of ammonium sulphate per litre in excess of this) a perfectly 

 pure preparation is obtained. 



Considerable difficulty has been experienced in obtaining serum- 

 albumin in a crystalline form. Formerly it was obtained almost 

 entirely from the blood of the horse, but even here the attempt to 

 obtain a crystalline preparation does not always meet with success. 

 In these cases Giirber's method was employed. The serum was 

 mixed with an equal volume of concentrated ammonium sulphate 

 solution ; then, to the filtrate from the precipitated globulin, ammo- 

 nium sulphate solution was added until there was an incipient 

 turbidity; on allowing the mixture to stand the serum-albumin 

 crystals separated. Inagaki has recently shown that crystalline 

 serum-albumin can be readily obtained by the Hopkins and Pinkus' 

 method, i.e. t in the presence of free acid. Crystallisation also takes 

 place more readily at a somewhat higher temperature (35-4O). The 



