VOL. 4 (1950) DPN AND GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE 



161 



EXPERIMENTAL 



The enzyme was prepared as previously described^ and recrystallized four times. An aliquot of 

 the crystal suspension in ammonium sulphate was centrifuged at about loooo rpm, drained, and 

 dissolved in 0.03 M sodium pyTophosphate — 0.003 ^I cysteine buffer at pH 8.3. This enzyme solution 

 was prepared fresh for each experiment. The composition of reaction mixtures is given in the tables. 



THE DISSOCIATION CONSTANT OF ENZYME AND BOUND DPN 



The enzyme and bound DPN concentrations cannot be varied independently 

 unless one resorts to partial removal of DPN with norit. The latter procedure introduces 

 additional variables due to the instability of the DPN-free enzyme and so a dilution 

 method was employed. It was possible to follow the reactions in the more dilute solu- 

 tions by using cuvettes with a longer light path. 



The experiment consisted in comparing the rates of reaction in two solutions 

 identical in all concentrations except that of the enzyme-DPN complex. The results of 

 such an experiment are described in Table I. It may be seen that the directly measured 



TABLE I 



THE DISSOCIATION OF ENZYME AND "BOUND" DPN 



Two reaction mixtures were prepared, one with a total volume of 6 ml and the other of 30 ml. The 

 former was in a cell of 2 cm and the latter in a cell of 10 cm length. Both reaction mixtures contained 

 in moles per ml, 6-io-® arsenate, 3-10-^ cysteine, 5-10-^ pyrophosphate (pn 8) and 2-10-® dl- 

 glyceraldehyde (the latter added to start the reaction). The two reaction mixtures differed however 

 in that the 2 cm cell contained 1.77- 10-® and the 10 cm cell 3.54- 10-^ M per ml of enzyme - DPN. 



* After addition of glyceraldehyde phosphate. 



rates were identical. This means that the decrease in rate due to the 5-fold dilution 

 of enzyme-DPN complex was exactly compensated by the 5-fold increase in light path. 

 Since the observed rate was proportional to the concentration of undissociated enzyme- 

 DPN, it follows that no measurable increase in dissociation occurred on dilution. In 

 order for this condition to hold, it would be necessary for the dissociation constant of 

 enzyme-DPN to be of the order of i-io"^" M/ml or less. Since in fact no evidence of 

 dissociation was obtained at all in this experiment, the above figure may be considered 

 only to be an upper limit*. An analogous dilution experiment with a small amount of 

 enzyme and added DPN with glyceraldehyde phosphate as substrate showed a change 



* In work which will be reported in detail at a later date it has been shown that bound DPN 

 equilibrates rapidly with radioactive DPN labelled with P^^ This is in harmony with the conclusion 

 that the bond between DPN and enzyme is not of the covalent type and that the bound DPN ex- 

 hibits a finite dissociation. 



References p. i6g. 



11 



