472 VITAMIN Bi2 



ml. of 0.1 M phosphate-citrate buffer which contained 0.1 % sodium bisul- 

 fite (met a). This was also superior to autoclaving for the same length of 

 time with distilled water. Harrison et al}"^ used with the E. coli cup-plate 

 method a steaming period of 30 minutes at pH 5 to 6 in the presence of 

 potassium cyanide. The presence of a small amount of cyanide has been 

 reported by Wijmenga et al.^^ to facilitate the release of vitamin B12 forms 

 from natural materials. 



Hot-water extraction in all reports releases about 80% or more of the 

 vitamin B12 activity. It has not been determined whether the added incre- 

 ment resulting from the proteolytic action of crude enzyme preparations 

 is due to vitamin B12 itself or to desoxyribosides. It is quite possible that 

 the "battery" of enzymes in the various crude preparations would release 

 many of the active soluble fragments of desoxynucleic acid or intact desoxy- 

 nucleic acid which is also active. Furthermore, vitamin B]2 activity has 

 been shown to be synthesized by contaminating microorganisms in digests 

 even in the presence of toluene, ^^ and enzyme preparations contribute a 

 significant amount of vitamin Bi2.^^ It appears that release with enzymes 

 which require 1 to 2 days should not be used until further study indicates 

 a definite superiority. On the basis of present data autoclaving for 5 minutes 

 or steaming for 30 minutes with phosphate, acetate, or citrate buffer be- 

 tween pH 5 and 6 will give satisfactory release of vitamin B12. The use of 

 cyanide or sulfite should be further investigated. 



VIII. standardization of Activity 



THOMAS H. JUKES and WILLIAM L. WILLIAMS 



The official U.S. P. qualitative and quantitative procedure for estimation 

 of pure crystalline vitamin B12 is as follows '} 



Vitamin B12 is a cobalt-containing substance usually produced by the 

 growth of suitable microbial organisms, or obtained from liver. "\A^en as- 

 sayed by the method described below, it has a purity of not less than 95 %, 

 calculated on the anhydrous basis. 



Description. Vitamin Bxo occurs as dark-red crystals or as a crystalline 

 powder. The anhydrous compound is very hygroscopic and when exposed 

 to air it may absorb about 12 % of water. 



64 H. G. Wijmenga, W. L. C. Veer, and J. Lens, Biochim. et Biophys. Acta 6, 229 



(1950). 

 •^^ D. V. Tappan, U. J. Lewis, and C. A. Elvehjem, .46s/;-. Papers 119th Meeting, 



Am. Chem. Soc, p. 20A, 1951. 

 ^ U. S. Pharmacopeia, 14th revision, p. 660, 1950. 



