II. CHEMISTRY 529 



factor) was recognized, isolation of vitamin H was attempted from its 

 most potent sources, i.e., liver and yeast, and no attention was given to 

 egg yolk as starting material. It has been shown that vitamin H as it occurs 

 in yeast and liver could not be dissolved in water or in any other solvent, 

 at least not in suthcient quantity to be effective in the prevention of egg 

 white injury in rats.''^ Autolysis of yeast in the presence of toluene led to 

 liberation of vitamin II in a water-soluble form. Chloroform, on the other 

 hand, inhibited reversibly this apparently fermentative reaction. On the 

 basis of this observation the conclusion was drawn that biotin (vitamin H) 

 is present in food products such as yeast and liver, mainly in bound form, 

 and may be liberated, at least from yeast, by fermentative action. 



In view of the fact that liver was found to be a more potent source of 

 the curative factor of egg white injury than is yeast, ^^ the question arose 

 as to whether or not vitamin 11 (biotin) could be separated also by autolysis 

 of liver tissue from the water-insoluble complex in which it occurs. Several 

 attempts to do this under varying experimental conditions and again using 

 animal bioassays for vitamin H failed.'^ In contrast, digestion with pepsin 

 and even more with papain, ^^"^^ but not with trypsin, or autoclaving at 

 high pressure, ^^ with or without the addition of acid, have proved satis- 

 factory procedures for the liberation of vitamin H (biotin) from a dry liver 

 powder. 



In these studies only animal assays were used. After the identification 

 of biotin with ^'itamin PI, microbiological test methods made the study of 

 bound and free biotin easier and more accessible to a quantitative and 

 better controlled analysis. With the more sensitive microbiological assay 

 Snell et al}^ found that autolysis increased the amount of biotin that could 

 be extracted from liver, whereas in animal assays using egg white injury as 

 test object such release of biotin from bound form was not demonstrable.'^ 

 Acid or enzymatic hydrolysis gave maximum yields of biotin from a variety 

 of natural materials, not only in microbiological tests^""^^ but also in animal 

 assays.^® 



Before the identification of vitamin H with biotin the first isolation of 

 vitamin H was attempted from the alcohol-insoluble fraction of beef liver 



'* P. Gyorgjs J. Biol. Chem. 131, 73.3 (1939). 



•6 P. Gyorgv, R. Kuhn, and E. Lederer, J. Biol. Chem. 131, 745 (1939). 



'' J. G. Lease and H. T. Parson.s, Proc. Am. Soc. Biol. Chem., J . Biol. Chem. 105, 1 



(1934). 

 '* J. G. Lease, Z. Viiaminforsch. 5, 110 (1936). 



'9 E. E. Snell, R. E. Eakin, and R. J. Williams, J. Am. Chem. Soc. 62, 175 (1940). 

 "V. H. Cheldelin, M. A. Eppwright, E. E. Snell, and B. M. Guirard, Univ. Texas 



Publ. 4237, 15 (1942). 

 «' R. C. Thompson, R. E. Eakin, and R. J. Williams, Science 94, 589 (1941). 

 22 J. O. Lampen, G. P. Bahler, and W. H. Peterson, .7. Nulnlion 23, 11 (1942). 



