548 THE BIOCHEMISTRY OF B VITAMINS 



developed; they depend upon the destruction of biotin by Raney's 

 nickel 35 and by oxidation with permanganate. 30 The oxygen analogue is 

 not appreciably affected by either of the reagents, but Raney's nickel 

 quantitatively converts biotin to desthiobiotin and permanganate oxidizes 

 biotin to the corresponding sulfone. Since neither desthiobiotin nor the 

 sulfone of biotin possesses any appreciable activity for Lactobacillus 

 arabinosus, as well as for several other organisms, such organisms which 

 respond to oxybiotin may then be utilized for a direct assay for the 

 oxygen analogue. 



Indirect assays for oxybiotin by assaying directly for biotin in the 

 presence of oxybiotin have been developed. The response of Lactobacillus 

 arabinosus to moderate amounts of oxybiotin is prevented by either 800 

 my per 10 cc of biotin sulfone or 70 y per 10 cc of y- (3,4-ureylenecyclo- 

 hexyl) butyric acid. 37 Under these conditions, neither of the inhibitors 

 appreciably affects the utilization of biotin by the organism. The biotin 

 is thus determined directly, and from the response of the organism to 

 the assay sample in the absence of the inhibitors a differential assay for 

 oxybiotin is obtained. Of course, large amounts of oxybiotin overcome 

 the toxicity of the inhibitors and would prevent the determination of 

 relatively small amounts of biotin. 



Another indirect assay depends upon the inability of Streptococcus 

 jaecalis R to utilize the oxygen analogue effectively. This permits the 

 estimation of biotin in an extract with only slight interference from the 

 oxygen analogue. By simultaneous assays with Lactobacillus arabinosus 

 and Streptococcus jaecalis R, a direct assay for biotin and a differential 

 assay for the oxygen analogue of biotin have been developed. 32 



With these assays, oxybiotin has been demonstrated not to occur 

 naturally in any of the organisms tested, and it has been possible to show 

 that the analogue is utilized as such without prior conversion to biotin. 

 Thus, permanganate destroys the biotin activity of hydrolysates of cells 

 of either Saccharomyces cerevisiae or Rhizobium trifolii grown in a biotin- 

 containing medium, but does not destroy the biotin-like activity of such 

 hydrolysates from cells of the organism grown in the presence of oxy- 

 biotin instead of biotin. 36 In balance experiments 38 with Saccharomyces 

 cerevisiae 139 grown in a medium containing 10 my of DL-oxybiotin 

 per 250 cc, essentially all (94-97 per cent of the compound) was recovered 

 from the medium and cells. At higher concentrations (100 my per 250 cc), 

 the recovery was lower (84 per cent) . By three different methods, the 

 Raney's nickel method, the permanganate method, and the differential 

 growth inhibitor method, it was demonstrated that oxybiotin alone 

 (with the exception of the small amount of biotin added with the in- 

 oculum) accounted for all the biotin-like activity in the cells. 38 



