l)lSCUSS/0\ 311 



is thill with respect to tlic mechanism in the bacterial limiinescent system, 

 1 dont have any e\ icience whether one molecule ol FMN is reduced or 

 whether two molecules of FMN are reduced on the en/yme surface. I think 

 you can write reaction mechanisms for either one or the otlier. Perhaps Dr. 

 .McKlroy has some evidence that there are two. but if he does, I would like 

 to know. 



Dr. Mc:Elrc)v: The only evidence we have is purelv kinetics, i.e., we must 

 plot the square of the F'MNH:: concentration in the Lineweaver-Burk plot in 

 order to obtain a straight line. 



Dr. Cormikr: The evidence that we had of course for two flavins was th'.* 

 fact that you could isolate an enzyme in which FMN was bound to the 

 enzyme and that this did not produce light until one added DPNH. and 

 aldehyde, FMN, and DPNH oxidase. You had to add exogenous FMN to 

 do it. However, I never could find out whether it was necessary to have 

 only one or Ijoth molecules of FMN reduced prior to light emission. 



Dr. Chase: May I ask if there is anything in the structure of the Cypridina 

 luciferin which would react with cyanide? Cyanide combined apparently 

 \ery readily and irreversibly with luciferin so that you can't get light. 



Dr. White: Does it combine with luciferin or with the enzyme? 



Dr. McCapra: This is with the luciferin, not witfi the enzyme. Well, I 

 don't think that under neutral conditions cyanide adds in any specific way 

 to luciferin. I mean it has to be catalyzed some way. 



Dr. McElrov: There is an important point here, though, with regard to 

 the Apogon system. It seems like a possibility from Dr. Johnson's data that 

 the enzyme can lose a metal c]uite readily. If the metal isn't an absolute 

 requirement for luminescence then cyanide would have no inhibitory effect. 

 There would be an inhibitory effect only when you add the metal. This is 

 one difference between the Apogon luciferase and the Cypridina luciferase. 



Dr. Chase: I have learned through a personal communication from Dr. 

 Hirata tliat cyanicie has no effect at all on their crystalline Cypridina luciferin. 

 He didn't give any details. This is exactly contrary to the very definite 

 results that Dr. Giese and I got with cyanide. 



Dr. Chase: Richard J. Lederman, a student at Princeton, and I have 

 been studying the effects of certain factors on the activity of Cypridina 

 luciferase, measuring the luminescent reaction with a photoelectric recording 

 light integrator, and taking the first order reaction rate constant as repre- 

 senting the activity of the enzyme. 



In the course of this work it was found that the concentration of the 

 phosphate buffer which was used to stabilize the pW of the reaction mixture 

 had a considerable effect ujjon the enzyme activity. Ordinarily one uses a 

 phosphate concentration of about 0.05 M for the luminescent reaction of 

 Cypridina luciferase and luciferin. We found that if the phosphate buffer 

 concentration was increased to 0.3 M the rate constant was reduced to about 

 one-half, whereas when phosphate concentration was lowered to about 0.01 M, 



