On the Chemistry of Light Production in Luminous Organisms. 89 
luciferin which is boiled is very quickly oxidized and converted into 
oxyluciferin. Acid retards and alkali favors the oxidation of the lucif- 
erin (apart from luciferase). A solution of luciferin made slightly acid 
with HCl may be boiled for 25 minutes in the air without complete 
oxidation, whereas a neutral or slightly alkaline solution is quickly 
oxidized. <A solution of luciferin will keep very well in a tall test-tube 
if the tube is left undisturbed. Diffusion of oxygen into the depths of 
the tube is very slow. Perhaps the best way to obtain a concentrated 
solution of luciferin is to filter the hot-water extract of Cypridina 
directly into a tall, narrow vessel and pass a current of CO, through it 
while cooling. The slight acidity and the anaerobic conditions both 
prevent oxidation. A little dilute acetic acid may be used in place of 
carbon dioxide. The oxidation product of luciferin, oxyluciferin, must 
also be present in crude luciferin solution to a greater or less extent, 
depending on the amount of oxidation which has taken place. Al- 
though both luciferin and oxyluciferin will pass collodion or parchment 
membranes, I have been unable to obtain them in crystalline form and 
presume that they also are present in the colloidal state. 
With these preliminary remarks on the preparation of impure lucif- 
erase and luciferin solutions, let us examine the chemical and physical 
characteristics of the two substances. Two points must be borne in 
mind. First, the wonderful delicacy of the luciferin-luciferase reac- 
tion. One part of luciferase in about 1,700,000,000 parts water will 
give a visible light on the addition of luciferin, and vice versa. A pre- 
cipitant must therefore precipitate absolutely; otherwise a mere trace 
of unprecipitated photogenic substance could be detected. It is also 
very necessary to make sure that in the vessel used in testing the pre- 
cipitation none of the photogenic solution is adherent to the sides in 
such a position as to escape contact with the precipitant. 
While a quantitative determination of the amount of luciferase (or 
luciferin) remaining in solution after adding a precipitant could be 
worked out by a method of dilution, 7. e., by determining how much 
water it would be necessary to add in order that the diluted solution 
would fail to give light with luciferin (or luciferase), this would be a 
tedious procedure. A rough estimate of the luciferin or luciferase in 
solution can be obtained by the brightness and duration of the lumin- 
escence, if one keeps in mind the following points: First, the greater 
the concentration of luciferin or luciferase the brighter the light. If 
luciferin is concentrated and luciferase is diluted the light will last 
a long time, while if luciferase is concentrated and luciferin weak a 
flash of light will occur which disappears very quickly. Bearing this 
in mind, one can judge fairly well whether precipitation has occurred 
and if it is only partial or very nearly complete. 
Second, both luciferase and luciferin are adsorbed by many sub- 
stances in a finely divided state—for instance, animal charcoal, freshly 
