290 



LIGHT AND LIFE 



the decay rate is not nearly so rapid. This observation and the fact 

 that, under these conditions, a doubling of the preincubation time 

 essentially doubles the steady state value suggest that an intermediate 

 is not only formed, but is also slowly accumulated. 



The intermediate is relatively heat-stable. If, for example, a stand- 

 ard assay system is preincubated for 30 minutes under anaerobic 

 conditions, heated at 100°C for 1^2 minutes (without prior flushing 

 with oxygen) , and then cooled to room temperature, an intermediate 



6000- 



10 20 30 



seconds 



10 20 30^^ I 



seconds 



TIME 



5 10 



minutes 



15 



Fig. 9. Kflct t of anaerobic preincubation on the kinetics of the reaction. 



can be demonstrated in the heated extract. Fig. 10 shows that the 

 addition of a portion of such a heated extract to a system containing 

 only crude huiferase, GSH, and phosphate buffer results in an al- 

 most instantaneous rise to a steady state. The kinetics of this re- 

 action is too fast to follow with the recorder tised (relaxation time 

 = 1 second fidl scale) . Thus the limitations of the recorder make 

 kinetic measurements uncertain for time periods of less than 1 second. 

 No light is obtained above background by flushing the preincubated 

 and heated extract with oxygen. Apparently the somewhat slower 

 kinetics observed in Fig. 9 are tiue to the limiting rate at which 

 oxygen is dissolved in water under these conditions. 



