CHEMICAL CONSTITUTION 



trate prepared by high pressure hydrolysis of an alcohol-insoluble 

 fraction of beef liver. Inert material was precipitated with alcohol 

 and acetone, and an active fraction was precipitated by means of 

 phosphotungstic acid, and the precipitate was decomposed with 

 barium hydroxide. Pure biotin ester was prepared from this con- 

 centrate by esterification with methanolic hydrogen chloride, followed 

 by two chromatographic adsorptions on Brockmann alumina. The 

 yield from 5 litres of liver concentrate was 70 mg. F. Kogl and E. J. 

 ten Ham ^ prepared j3-biotin from a liver concentrate by chromato- 

 graphic adsorption on norit, elution with ammoniacal acetone, esteri- 

 fication, chromatographic adsorption of the ester from chloroform on 

 alumina and elution with a mixture (9 : i) of acetone and methanol, 

 followed by re-adsorption on alumina from acetone, elution with a mix- 

 ture of acetone and methanol and, finally, hydrolysis to the free acid. 

 Biotin was also prepared from milk. A concentrate was esterified 

 with acid methanol, and the ester was chromatographed first on 

 Decalso and then on activated alumina.® The crude crystalline ester 

 obtained on elution was purified by washing with ethyl acetate, sub- 

 limation in vacuo and re-crystallisation from a mixture of ether and 

 methanol. The free biotin obtained on hydrolysis was identical with 

 the biotin from liver. A yield of 25 to 40 % was obtained. 



References to Section 2 



1. F. Kogl and B. Tonnis, Z. physiol. Chem., 1936, 242, 43. 



2. F. Kogl and L. Pons, ihid., 1941, 269, 61. 



3. P. Gyorgy, R. Kuhn and E. Lederer, /. Biol. Chem., 1939, 131, 745. 



4. V. du Vigneaud, K. Hofmann, D. B. Melville and P. Gyorgy, 



ibid., 1941, 140, 643. 



5. F. Kogl and E. J. ten Ham, Z. physiol. Chem., 1943, 279, 140. 



6. D. B. Melville, K. Hofmann, E. Hague and V. du Vigneaud, /. 



Biol. Chem., 1942, 142, 615. 



3. CHEMICAL CONSTITUTION OF BIOTIN 



Chemical Constitution of p-Biotin 



^-Biotin methyl ester, m.p. 166 to 167° C, has the empirical 

 formula, CnHigOgNaS,^ and free j8-biotin, m.p. 230 to 232° C, has the 

 formula, CioHig03N2S.^ 



Biotin is a carboxylic acid and, on treatment with alkali,^ it 

 gave a sulphur-containing diamino-carboxylic acid, CgHigOgNgS (H), 

 containing one CO group less than biotin. This was interpreted as 

 indicating the presence of a NN' -substituted cyclic urea group. On 

 oxidation with hydrogen peroxide, it yielded a sulphone (HI), sug- 

 gesting that the sulphur was present as a thio-ether linkage. F. Kogl 



407 



