Biotin in Elementary Bodies of Vaccinia. ∗

Studies of the constituents of elementary bodies of vaccinia have shown that preparations purified by a standard technic exhibit uniformity in their chemical constitution and in their biological activity. 1 , 2 The virus appears exceedingly complex and therefore the view that respiratory catalysts, which are known to play an important rôle in bacterial metabolism, may function in the organization of elementary bodies of vaccinia is not untenable. With this in mind, we have examined elementary bodies for growth-factors.

By means of a technic similar to that described by McDaniel, Woolley, and Peterson, 3 vaccine-virus has been shown to contain relatively large quantities of a substance capable of stimulating the growth of Clostridium butylicum in a synthetic medium. Moreover, partial hydrolysis of the virus by normal alkali or 4 normal sulfuric acid gives rise to increased quantities of this substance in active form, which, on the basis of studies similar to those made by Snell, Eakins, and Williams, 4 and others 5 , 6 on liver, yeast, egg-yolk, etc., we believe to be biotin.

Two recognized technics exist for the microbiological assay of biotin. The first, employed by Snell, Eakins, and Williams 4 depends upon the growth-stimulus afforded by this substance to a standardized strain of yeast, while the second depends upon the stimulus afforded to the growth of Clostridium butylicum in a synthetic medium devoid of biotin. 3 Since the growth-requirements are simpler and somewhat better understood in the case of Clostridium butylicum, the second technic was adopted as affording the greatest guarantee against introducing with the material to be tested other physiologically active substances.

Elementary bodies of vaccinia were prepared for biotin-assay in 3 ways: first, as dried virus, resuspended by physical agitation; second, by dissolving the dry virus in normal alkali with the aid of heat; and third, by refluxing the dry virus with 4 normal sulfuric acid for 30 minutes at 100°C.