Alternative Pathways of Electron Transport 195 



tained only small traces of this enzyme which for special 

 reasons henceforth shall be called vitamin K reductase. In 

 fact, we met with great difficulty in trying to enrich and pre- 

 pare the substance in a pure state, and finally attained our 

 goal only by combining several efficient methods which have 

 recently been developed for the separation of proteins. 

 Starting with extracts of beef liver, which is the best source 

 of this enzyme, an overall purification of more than 4,000-fold 

 had to be performed before an electrophoretically pure pre- 

 paration was obtained. To our great surprise the new enzyme 

 proved to be a hitherto unknown flavoprotein (Martins and 

 Marki, 1957). The spectrum shows two peaks in the visible 

 region, one at 380 mpi, the other at 456 m{j, and a shoulder at 

 485 mpL. The normal peak is found in the u.v. region at 273 my.. 

 The prosthetic group of the enzyme proved to be flavin- 

 adenine dinucleotide (FAD). The coenzyme can be reversibly 

 split off by dialysis against distilled water. The activity can 

 be fully restored by addition of a solution of authentic FAD. 

 The identity of the coenzyme was further checked by means 

 of the D-amino acid oxidase test for FAD, which was positive. 

 There are remarkable differences in the enzymic behaviour of 

 vitamin K reductase and cytochrome c reductase. Both can 

 react in the reduced state with phylloquinone, but only the 

 latter with cytochrome c. p-Chloromercuribenzoate (PCMBA) 

 in a concentration of 10 ~* m completely inhibits the Mahler 

 enzyme whereas vitamin K reductase remains absolutely 

 unaffected by the same inhibitor. Apparently the latter does 

 not contain enzymically active SH-groups ; on the other hand, 

 it is inhibited by low concentrations of dicoumarol and other 

 vitamin K antimetabolites. With 10"^ dicoumarol the inhibi- 

 tion rate is 100 per cent, with 10-^ m it is 95 per cent, and 

 even when a 10"'^ m concentration was used about 50 per cent 

 inhibition was still observed. Thus, dicoumarol is a very 

 potent inhibitor, the inhibition being of the competitive type. 

 Substances which are related chemically or in other respects, 

 e.g. dinitrophenol (DNP), antimycin, or hydroxynaphtho- 

 quinone derivatives like SN5949, are, however, without any 



