398 VITAMIN Bi2 



the use of other solvent mixtures such as n-or isopropanol containing 10 

 to 20 % water ; the separation of pink zones was satisfactory but little su- 

 perior to the separation with wet butanol. The pink-zone material was 

 further fractionated by repetition of partition chromatography and by 

 fractional precipitation with ammonium sulfate. 



For other partition chromatography, starch could be used instead of 

 silica. On starch columns, the pink fractions from both proteolyzed and 

 non-proteolyzed liver extracts separated into two pink zones with Rj values 

 of about 0.5 and 1.2, respectively. Non-proteolyzed liver gave mainly the 

 slow-moving component; the faster moving fraction was apparently the 

 product of autolysis of the liver extracts. 



Treatment of the pink-zone fractions with trypsin, followed by chroma- 

 tography on silica or charcoal, gave further purification, and then crystal- 

 lization from aqueous acetone was sometimes possible. It was found ad- 

 vantageous to precipitate the red pigment with phosphotungstic acid, before 

 final crystallization from aqueous acetone.'^ 



It was observed^ that purification of vitamin B]2 from liver extracts was 

 aided by n-butanol extraction of aqueous solutions containing fairly high 

 concentrations of ammonium sulfate. Chromatography with bentonite or 

 aluminum silicate columns was then used for final stages of purification. 

 The sharply defined pink bands were eluted, and these fractions were found 

 to crystallize from aqueous acetone. 



Investigation of other sources of vitamin B12 led to the discovery that it 

 is present in numerous natural materials, and particular interest is attached 

 to fermentation broths.^ The culture broths of strains of M ycohacterium 

 smegmatis, Lactobacillus arabinosiis, and Bacillus suhfilis, and of several 

 Streptomyces species such as S. roseochromogenus, S. griseus, and S. anti- 

 hioticus, have all been found to show vitamin Bi2 activity. From one of 

 these culture broths, produced by a strain of S. griseus, crystalline vitamin 

 B12 was isolated.^ 



In the course of work on the isolation of vitamin B12 from natural soiu'ces, 

 recourse has been made to paper strip chromatography. A novel method has 

 been devised^" in which concentrates containing vitamin B12 are subjected 

 to partition chromatography on paper strips developed with water-satu- 

 rated n-butanol. The developed strip is then dried and applied to the sur- 

 face of a plate of nutrient agar seeded with L. lactis Dorner for about 10 

 minutes. Inculcation of the plate overnight leads to a pattern of growth with 

 "zones of exhibition," which indicate the presence of active constituents. 



8 B. Ellis, V. Petrow, and G. F. Snook, J. Pharm. Pharmacol. 1, 60 (1949). 



8 E. L. Rickes, N. G. Brink, F. R. Koniuszy, T. R. Wood, and K. Folkers, Science 



108, 634 (1948). 

 '» W. F. J. Cuthhertson and K. L. Smith, Hiochcm. J. 44, V (1949) ; K. L. Smith, W. F. 



J. Cuthbertson, A. Walker, and K. A. Lees, Federation Proc. 9, No. 1, 230 (1950). 



