G. ROBERT GREENBERG 



an excellent high positive-potential acceptor from pyridine nu- 

 cleotides, but cytochrome c is reduced nonenzymatically by va- 

 rious compounds. For example, it is readily reduced by tetra- 

 hydrofolic acid. Potassium ferricyanide possesses a high posi- 

 tive potential and its reduction by DPNH can be followed by a 

 drop in its absorption at 420 mju. This compound may, how- 

 ever, oxidize substances other than DPNH. 



With crude systems one cannot be sure that the reduction of 

 pyridine nucleotides is coupled to the system being studied. 

 Thus in an extract of pigeon liver acetone powder after dialysis 

 there are substrate sources which bring about the reduction of 

 cytochrome c. Since these sources appear to possess rather 

 negative E'q values, all other conditions being the same, they can 

 play the major role in cytochrome c reduction. Thus reduced 

 pyridine nucleotide derived in a reaction mixture may be as- 

 sociated with the oxidation of only a portion of the hydrogen 

 donor substrate. In the end it may be necessary to make a 

 partial fractionation of the system using other assay methods in 

 order to remove some of these interfering reactions. 



Consideration may be given here to the effect that a coupled 

 system has on a multi-enzyme reaction sequence. On first 

 glance a coupled DPN-regenerating reaction may be thought of 



