232 BIOCHEMISTRY OF BACTERIAL LUMINESCENCE 



reach one-half of the steady-state value when pressure was released 

 was similar to if not identical with the time required for the lumines- 

 cence to reach one-half its maximum rate when reduced DPN was the 

 last component added. Thus it seems clear that the reaction which is 

 inhibited by pressure is the DPNH2-FMN reaction and that flavin 

 oxidation via the luminescent pathway is potentiated by pressure. The 

 gradual decrease in luminescence after the initial spike when pres- 

 sure is applied, can be viewed as the decrease in the reduced flavin 

 mononucleotide pool size and the converse effect, the slow rise fol- 

 lowing the "black out," is apparently due to the rise in the size of 

 this same pool as a consequence of the greater rate of the DPNH2- 

 flavin reaction when the pressure is released. 



The simplest interpretation of the observed effects is that (1) the 

 reduction of flavin by reduced DPN proceeds with a volume increase 

 on activation and that the application of high hydrostatic pressure 

 retards this process, while (2) the oxidation of flavin in the lumines- 

 cent reaction proceeds with a volume decrease on activation and is 

 thus accelerated under pressure. 



It should be pointed out that these are not the exclusive possible 

 interpretations of the observed effects. Although the effect of pressure 

 on the luminous oxidation of flavin is nearly instantaneous, at low 

 temperatures it is clear that there is considerable delay in reaching 

 the small spike maximum. If the effect were on the final reaction step, 

 it would seem unlikely that such a period of time should be re- 

 quired for the rate of the reaction to become maximal. Moreover, a 

 net increase in the reaction rate under pressure does not necessarily 

 mean that the activation step is pressure sensitive. Another possibility 

 is that some intermediate of flavin oxidation dissociates from an en- 

 zyme with a volume increase and further gives rise to a nonluminous 

 reaction. Under these conditions the application of pressure would 

 prevent the intermediate from being dissociated from the enzyme, 

 raise its concentration and result in the increased luminescence ob- 

 served. The same type of argument may be applied to the observed 

 effect on the DPNHo-FMN reaction in which it might be argued that 

 the association constants of one of the reactants might be appreciably 

 modified by pressure. If this is the meaning of the observation, the 

 association of the substrate with the enzyme would involve a net vol- 



