THE FOLIC ACID COMPLEX 



factor with methanol. No further purification was effected by pre- 

 cipitation with heavy metals or by fractional precipitation from a 

 concentrated aqueous solution. 



The method used in isolating the pure crystalline liver L. casei 

 factor was described in detail by Stokstad et al.^ A liver extract, 

 prepared by precipitating an aqueous extract of liver with alcohol, 

 was used as the starting material. It was adjusted to pH 8*5 and 

 heated to coagulate impurities, and the filtrate was acidified to ^H 3 

 and stirred with norit. The adsorbate was eluted with 0-5 N-ammonia 

 in 60 % ethanol at 70° C, and the eluate was acidified to pR 1-3 and 

 adsorbed on a column of Superfiltrol, which was eluted with 0-5 N- 

 ammonia in 60 % ethanol. The eluate was concentrated and neutral- 

 ised to pH 7-0, and the L. casei factor was precipitated as the barium 

 salt by adding alcohol and barium chloride. The barium salt was 

 dissolved in 0-2 N-methanolic hydrogen chloride, and the solution 

 left at 25° C. for an hour to convert the factor into its methyl ester. 

 The mixture was evaporated to dryness, the residue was dissolved in 

 water, acidified to pYi 6 to y and extracted with w-butanol. The 

 solution was chromatographed on a column of Superfiltrol, which was 

 washed with 92-5 % acetone and then eluted with 75 % acetone. 

 The ester was precipitated several times from hot aqueous methanol un- 

 til pure, and then hydrolysed with alkali to give the pure L. casei factor. 



The fermentation L. casei factor was produced by aerobic fermen- 

 tation with an unidentified bacterium belonging to the genus Coryne- 

 bacterium. The fermentation liquor, which contained 3 to 5 /xg. per 

 ml.,^ was acidified to pH 3-0 and stirred with norit. The adsorbate 

 was washed with water and 50 % ethanol and eluted with aqueous 

 alcoholic ammonia at 70° C. The eluate was neutralised and the 

 barium salt of the L. casei factor was precipitated by adding alcohol 

 and barium chloride solution. The barium salt was dissolved in 

 methanolic hydrogen chloride and allowed to stand in order to convert 

 the factor into its methyl ester. The mixture was neutralised and 

 evaporated to dryness and the residue dissolved in water. The 

 solution was extracted with n-butanol, and the extract evaporated to 

 dryness. The residue was dissolved in hot methanol and precipitated 

 by cooling, the process being repeated with modifications several 

 times until pure. The purified ester was then hydrolysed with baryta 

 solution and the hydrolysate treated with Florisil to remove impurities, 

 and then with barium chloride and alcohol to precipitate the barium 

 salt of the factor. This was dissolved in water, hydrochloric acid 

 was added, and the solution was cooled to 0° C. The precipitate was 

 re-dissolved in acidulated water containing a little calcium or sodium 

 chloride and the solution was cooled, giving a crystalline precipitate 

 of the pure L. casei factor. 



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