200 CARNEGIE INSTITUTION OF WASHINGTON. 



We may therefore conclude that if any temperature change occurs during 

 the kmiinescence reaction it is certainly less than 0.001° C, and probably less 

 than 0.0005° C, too small to be measured by this method. 



To prepare the luciferin solution, 2 grams of dried Cypridina were dissolved 

 in 20 c.c. hot water and 10 c.c. of this 10 per cent solution were used in the 

 thermos bottle in the above experiments. If we assume that 1 per cent of the 

 dried Cypridina is luciferin, 0.1 gram of luciferin on oxidation was not able to 

 raise the temperature of the 10 c.c. (in reahty 11 c.c, since 1 c.c. luciferase 

 solution was mixed with the 10 c.c. luciferin) of solution 0.001° C. This 

 means that 1 gram luciferin hberates at least less than 0.1 calorie during the 

 luminescence accompanying oxidation. 



It is because of the small energy change during oxidation of luciferin that 

 the reaction may be so easily reversed and oxyluciferin reduced. Most of the 

 reducing methods described in my last report involve reduction in acid solu- 

 tion or in a solution which becomes acid. Indeed, acid alone will cause a 

 slight reduction, and this is a function of the H-ion concentration, since any 

 acid added to oxyluciferin will cause a slight reduction to luciferin. The 

 change begins when the solution is about neutral, Ph = 7.1. Acid is not essen- 

 tial for reduction, however, as reduction can occur in alkaline solutions which 

 generate nascent hydrogen, as on addition of Al and NaOH, or merely on 

 mixing oxyluciferin with finely divided Al, Zn, or Mg, or in the presence of 

 (NH4)2S. . . .,, 



Since all the reducing methods which may be used with oxyluciferin will 

 also reduce methylene blue to its leucobase, I believe we may provisionally 

 use tliis reaction as a type to explain what happens when luciferin is oxidized. 

 As methylene blue contains no oxygen, its reduction consists in the addition 

 of 2 atoms of hydrogen. When leuco-methylene blue oxidizes, which it does 

 spontaneously in air, water is formed by the union of these 2 atoms of hydro- 

 gen with oxygen, thus: 

 CieHaoNs SCI (leuco-methelene blue) +0^=>Ci6 His N3 SCI (methylene blue) 



+H2O 

 For short, 



MHa+O^Z^M+HaO 



Writing the luminescent reaction in a similar way, we have : 



luciferin + 0:5Z±oxy luciferin + H2O. 

 For short, 



LH2-1-0^=±L-FH20. 



If we assume that the LH2 (luciferin) compound is dissociated to even the 

 slightest extent into L and hydrogen, adding the hydrogen ion will shift the 

 equilibrium toward the formation of that substance which involves the taking 

 up of hydrogen. Consequently, we may obtain a partial formation of luci- 

 ferin by adding an acid to oxyluciferin. Reduction of the H-ion concentra- 

 tion tends to shift the equihbrium in the opposite direction. Consequently, 

 addition of alkali favors the oxidation of luciferin, and it is quite generally 

 true that biological oxidations are favored by an alkahne reaction. In addi- 

 tion, oxygen in alkaline medium has a higher oxidation potential than in 

 neutral or acid media. I believe that this is the explanation of the action of 

 acid in formation of luciferin from oxyluciferin. 



Addition of acid is not the only means of favoring the formation of luciferin 

 from oxyluciferin. Any reaction which proceeds in one direction with evo- 

 lution of light should, theoretically, proceed in the opposite direction under 

 the influence of light. It is a fact that hght will cause the reduction of oxy- 

 luciferin. A tube of oxyluciferin exposed to sunlight for 6 hours or the mer- 

 cury arc for 2 hours will be partially converted into luciferin. It will lumi- 



