II. CHEMISTRY 397 



II. Chemistry 



DONALD E. WOLF and KARL FOLKERS 



The interest in an anti-pernicious anemia factor dates back to the dis- 

 covery^ in 192G that pernicious anemia could be controlled therapeutically 

 with whole liver. Soon after this epoch-making discovery, various groups of 

 in\-estigators began to fractionate liver extracts for the purification of the 

 anti-pernicious anemia principle. - 



A. ISOLATION 



The present review begins with a report^ in 1948 on the isolation of ^^- 

 tamin B12 as a red crystalline substance. The crystallization of vitamin B12 

 was the consummation of a long program of investigation. The crystalline 

 \'itamin B12 possessed a bioactix-ity^ • ^ for L. lactis Dorner of 11 X 10^ units 

 per milligram, and 0.000013 7 per milliliter of culture medium was capable 

 of supporting half -maximal growth under the conditions used. The new 

 vitamin was found to be clinically active when a single dose of 3 to 6 7 

 was injected intramuscularly in patients with pernicious anemia.^ 



The isolation of vitamin B12 was described later by several groups of 

 investigators. In each case, the isolation involved extensive use of various 

 types of chromatography. Liver extract®- "^ or the extract proteolyzed wath 

 papain was subjected to repeated adsorption on charcoal and elution with 

 hot 65 % alcohol. This tj^pe-step was followed by partition chromatography 

 on damp silica columns. The liver extractives were applied to the silica 

 columns in dry butanol, and the columns were developed with butanol 

 containing about 1 1 % water. Under these conditions, the partition chroma- 

 tograms readily showed three colored zones: (1) a fast-moving yellow zone 

 which could be washed from the column by continued development; (2) 

 a pink zone which moved slowlj^ down the column; and (3) a brown zone 

 which remained at the top of the column. By clinical trials, it was found 

 that nearly all the anti-pernicious anemia activity resided in the pink zone 

 which could be eluted with 50 % ethanol. Variations of this method involved 



' G. R. Minot and W. P. Murphy, J. Am. Med. Assoc. 87, 470 (1926). 

 - Y. SubbaRow, A. B. Hastings, and M. Elkin, Vitamins and Hormones 3, 237 (1945). 

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

 107, 396 (1948). 



* M. S. Shorb, Science 107, 397 (194S). 



* R. West, Science 107, 398 (1948). 



« E. L. Smith, Natm-e 161, 638 (1948); E. L. Smith, L. F. J. Parker, Proc. Biochcm. 



Sac. Biochem. J. 43, VIII (1948). 

 ' D. Hodgkin, M. W. Porter, and R. C. Spiller, Proc. Roy. Soc. (London) B136, 609 



(1949); K. n. Fantes, J. E. Page, L. F. J. Parker, and E. L. Smith, Prtjc. Roy. 



Soc. {London) B136, 592 (1949). 



