142 



THE CELL AND PROTOPLASM 



except for the protein component, wliicli 

 was not capable of synthesis. This was, 

 however, not correct. Four days after 

 Kuhn's work was published a work of mine 

 appeared showing that the prosthetic group 

 of the yellow ferment is not lactoflavin but 

 a lactoflavin-monophosphoric acid ester. 

 The discovery of the lactoflavinphosphate is 

 a case of good luck. It did not seem un- 

 reasonable to suppose that phosphoric acid 

 might be the intermediate link between 

 lactoflavin and protein. The only material 

 at my disposal at the time was a long since 

 spoiled solution of the prosthetic group of 

 the yellow ferment that had been split off 

 with methyl alcohol. Several weeks pre- 

 viously, however, when it was still fresh 

 the flavin concentration had been deter- 

 mined ; the phophorus could not, of course, 

 have disappeared. A microanalysis of the 

 phosphorus showed that the solution con- 

 tained the same concentration of phos- 

 phorus as there had been flavin at the begin- 

 ing. A new preparation of the prosthetic 

 group showed again a content of one atom 

 of phosphorus per molecule of flavin. But 

 it was necessary to prove that the phos- 

 phoric acid was really esterified with the 

 lactoflavin. This was a delicate task, as 

 all that remained of the prosthetic group 

 contained only about 50 mg. of lactoflavin. 

 The electrophoretic method previously used 

 enabled us to solve the problem. Lacto- 

 flavin in neutral solution is electrically neu- 

 tral and thus remains motionless on elec- 

 trophoresis. If now the prosthetic group 

 of the yellow ferment were esterified with 

 phosphoric acid, in neutral solution it 

 should migrate as an acid toward the anode. 

 It was even possible, on the basis of previous 

 studies on other phosphoric acid esters, to 

 predict the speed of migration that a lacto- 

 flavin monophosphoric acid ester should 

 show. The experiment was very exciting, 

 as the solution of the prosthetic group was 

 so weak that no color at all was visible. 

 Not until the experiment was finished could 

 the electrophoretic apparatus be placed be- 

 fore a mercury lamp. The yellow fluores- 

 cence, which is an extremely sensitive indi- 

 cator of flavins, showed that the prosthetic 



group had migrated towards the anode, 

 and even quantitatively at the calculated 

 speed. When the pure ferment was after- 

 ward produced on a larger scale, the cal- 

 cium salt of the lactoflavin phosphoric acid 

 was crystallized. 



The production in the pure state of the 

 yellow ferment was of a certain methodo- 

 logical interest, as it could be carried out 

 by the direct electrophoresis of a prepara- 

 tion containing only 3 per cent of the pure 

 ferment. For the production on a larger 

 scale, however, the electrophoretic purifi- 

 cation had to be supplemented with am- 

 monium sulphate fractionation. It was 

 found to be a flavoproteid.* On the as- 

 sumption that one molecule of the ferment 

 contained one molecule of the prosthetic 

 group, the riboflavinphosphate, the molec- 

 ular weight was calculated as 75,000; this 

 value was confirmed in Svedberg's institute 

 by means of ultracentrif ugation. The light 

 absorption curve of this "old" yellow fer- 

 ment is similar to that of the free flavin 

 (see Fig. 1). However, it should be noticed 

 that the absorption bands of the prosthetic 

 group are displaced about 20 mp towards 

 the red end of the spectrum when it is 

 linked to the protein to form the ferment. 

 This displacement is interesting because it 

 means that the activation energy of the 



Fig. la. Absorption spectrum of the "old" yel- 

 low ferment (TheorelD, 

 Wave length in mji 



4 Because of the high content of polysaccharides 

 in the crude preparations, it had previously been 

 considered that the ferment might be a flavopoly- 

 saccharide. 



