VOL. 4 (1950) DPN AND GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE 



163 



arsenate and then precipitating the enzyme with ammonium sulphate at a final concen- 

 tration of 85% saturation. It was found that 90% or more of the enzyme was precip- 

 itated and that the ratio of DPNH to protein in the precipitate was the same as that 

 of DPN to protein in the original solution. 



For the interpretation of reactions with added DPN an additional consideration 

 is important, namely, whether added DPN can displace DPNH at site I. From the fact 

 that DPN at site I is dissociable one would expect the same to hold for DPNH. The 

 problem of displacement would then be resolved by, a determination of the relative 

 dissociation constants of enzyme with DPN and DPNH. Theoretically this could be 

 done by determining the ratio of DPN to DPNH in the enzyme when enzyme-DPNH 

 is precipitated in the presence of added DPN. 



A preliminary experiment of this type is presented in Table HI ; it gives qualitative 

 evidence that displacement of DPNH by DPN does occur and that the dissociation 



TABLE III 



COMPETITION BETWEEN DPN AND DPNH 



DPN in enzyme was reduced by addition of arsenate and an equivalent amount of triosephosphate. 

 Aliquots of the reduced enzyme were treated as follows. In (A) 0.5 ml of enzyme containing 12.5 mg 

 of protein, -f- o.i ml of HgO, was precipitated with 3 ml of saturated ammonium sulphate. In (B) 0.5 ml 

 of enzyme + o.i ml of DPN solution (2.4-io— ' M) was incubated for 3 minutes before being pre- 

 cipitated with ammonium sulphate. The precipitates were separated by centrifugation at loooorpm 

 and dissolved in cysteine-pyrophosphate buffer. 



Calculated from optical density at 276 m/<. 

 Determined by biuret method. 

 *** An excess of glyceraldehyde phosphate was added in order to reduce DPN completely. 



constants of the oxidized and reduced forms with the enzyme are at least of the same 

 order of magnitude. The chief objection that might be raised is that the high concen- 

 tration of ammonium sulphate may change the equilibrium. 



An analysis of the experiment shows that although a stoichiometric amount of 

 glyceraldehyde phosphate was used, the reaction was only 70% complete when DPN 

 was added. This value is calculated from the additional DPNH which appeared when 

 excess triosephosphate and arsenate was added to the dissolved precipitate of the enzyme 

 in experiment A. Accordingly there must have been residual triosephosphate in B when 

 DPN was added. The excess DPN in B then drove the reaction to completion as shown 

 by the DPNH recoveries in A and B. 



Some of the DPNH in the supernatant fluid of B, therefore, arose by reduction of 

 added DPN and hence did not represent DPNH displaced from the enzyme. A rough 

 estimate of the amount actually displaced is (by comparison with experiments A) equal 

 References p. i6g. 



