SERUM GLOBULIN 41 



The following method of preparing serum globulin is based 

 upon the observation of Alexander Schmidt (38) and Kuhne (14) 

 that " insoluble" serum globulin can be precipitated from serum, 

 previously diluted with ten volumes of water, by the passage of 

 C0 2 . It appears to be difficult or impossible to bring about this 

 precipitation in the total absence of salts of stronger acids, e.g., 

 NaCl. I have repeatedly observed that if serum globulin, pre- 

 cipitated by C0 2 and carefully washed, be dissolved in a minimal 

 quantity of NaOH or KOH it cannot be reprecipitated by dilu- 

 tion and the passage of C02, although a distinctly acid and very 

 opaque solution results. 



Three litres of ox-serum are diluted with ten volumes of dis- 

 tilled water, and C0 2 is bubbled through it at a good rate for 

 about half an hour. The globulin which is thus precipitated 

 is allowed to settle in tall glass cylinders,* the supernatant 

 fluid being syphoned off after settling. The precipitate is then 

 washed with about 60 litres of distilled water, in two portions. 

 The globulin is then dissolved in a minimal quantity of 

 N / 10 HClf and immediately reprecipitated by cautious neutral- 

 ization with AT/10 KOH. This precipitate, after settling and de- 

 cantation of the supernatant fluid, is washed in 60 litres of distilled 



* For the settling and washing of protein precipitates I employ wide- 

 mouthed glass cylinders from 50 to 60 cm. high and about 10 cm. in diameter, 

 closed by ground glass stoppers. The syphon is provided with a side tube 

 through which it can be filled and can then be closed by a rubber tube provided 

 with a pinch-cock. When the protein is being washed with alcohol or with 

 ether, the syphon is filled with alcohol so as to avoid accidental contamination 

 of the contents of the cylinder with water. The syphon is supported on a 

 stand which is so arranged that the height of the opening of the syphon above 

 the precipitate can be adjusted by turning a screw. The greater part of the 

 liquid is first rapidly run off with the opening of the syphon a good way above 

 the precipitate. The syphon is then lowered and the remainder of the liquid 

 run off more slowly. 



f This can be calculated from the fact that 1 litre of serum yields about 

 5 grams of globulin and that about 20 X 10~ 6 gram equivalents of the strong 

 monobasic acids just suffice to dissolve one gram. If acetic acid be preferred, 

 about 100 X 10~ 5 equivalents are required to dissolve 1 gram (9). 



It has been shown by Hammarsten (7) that "insoluble" serum globulin 

 is not readily "denatured," i.e., altered in properties, by acids. "A solution 

 of serum globulin containing 0.2-0.3 per cent acetic acid remains unchanged 

 for days at a low temperature, and one can recover the serum globulin unal- 

 tered by neutralization." In the presence of stronger acids denaturation is a 

 matter of some hours. 



