302 RIBOFLAVIN 



connected with this disease. A lack of vitamin B2 , the heat-stable companion of the 

 heat-labile vitamin Bi , at that time was considered to be the cause of pellagra. In 

 the beginning of 1932, the author of this article started the isolation of so-called vita- 

 min B2 at Kuhn's institute. Gyorgy performed the biological tests on rats, according 

 to the method of Sherman and Bourquin; later on, when our preparations became 

 purer, the diet of the animals had to be modified somewhat, since it was lacking not 

 only in vitamin B2 but also in another member of the B-vitamin complex. 



The literature contained very little and vague data on the concentration of vi- 

 tamin B2 from yeast and liver, which turned out to be of little value for the pro- 

 cedure of isolation. For the adsorption of the vitamin, fuller's earth in acid solution 

 had been recommended. Another valuable adsorbent soon was found, which adsorbed 

 vitamin B2 already from neutral solution; this was "Frankonit KL" (a bleaching 

 earth produced by the Pfirschinger Mineralwerke, Kitzingen/Main, Franconia), 

 which since that time has been used frequently in biochemical work. Before the vita- 

 min B2 investigation, the existence of our sample of this adsorbent had been forgotten 

 in one of the laboratory closets, after I had tried it with little success for the poly- 

 merization of isoprene. 



None of the known methods was fit for elution of the vitamin from the adsorbent 

 in yields worth mentioning. But a wrong hypothesis about the chemical nature of 

 vitamin B2 helped me to find the right trail. In one paper of the literature it had been 

 assumed that vitamin B2 might contain iron. With regard to the biological properties 

 of hemin derivatives, one would have been inclined to guess that iron porphyrin 

 complexes were involved. Since pyridine is a solvent for compounds of this tj'pe, this 

 substance, diluted with water and alcohol, was tried out for the elution of vitamin B2 

 adsorbates. This attempt was a full success. The later progress of our investigation 

 made it clear that iron has nothing to do with vitamin Bo , but the wrong hypothesis 

 had proved useful. 



The successful elution drove the isolation procedure one essential step forward. It 

 soon became evident that all eluates, which were active in the animal experiments, 

 had a greenish-yellow color and showed a yellowish-green fluorescence in the light of 

 a quartz lamp. Therefore, the idea came up that vitamin B2 itself might be colored, 

 and the investigation was continued with attention to this assumption. 



It still was difficult to obtain purified preparations of the vitamin from extracts of 

 yeast, liver, heart, or kidnej^ because of the presence of large amounts of accom- 

 panying substances. An 80% methanol extract of egg white turned out to be a much 

 better starting material. The concentrated, greenish-yellow eluates on precipitation 

 with AgNOs gave a brownish-red silver salt of the vitamin. In later experiments, a 

 precipitation with TI2SO4 was inserted for further purification. This salt had been 

 chosen with regard to the chemical relationship of certain thallous and silver salts. 

 The first few milligrams of crystallized "ovoflavin" became available for analysis 

 shortly before Christmas, 1932. The animal tests proved without an.y doubt the 

 growth-promoting nature of the substance. 



At this stage of the investigation, we received knowledge that the pharmacologist 

 Ph. Ellinger in Diisseldorf, working on the fluorescence of animal organs, had pre- 

 pared a colored concentrate from skimmed milk, which obviously was similar to 

 ovoflavin. Our methods of purification proved to be particularly applicable to whey. 

 We therefore changed over to this starting material. It was, however, not possible to 

 handle the large amounts of liquid in the laboratory. Therefore, the first step of the 

 concentration, the adsorption, was carried through in a large cheese dairy in Bavaria. 

 With this procedure we socm were able to obtain 1 g. of crystallized "lactoflavin" 

 from 5400 1. of whey, thus opening a way for the elucidation of the chemical structure 

 of vitamin B2 . 



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