EXTRACTION WITH AQUEOUS SOLVENTS AND EFFECT OF OXYGEN.
If the luminous tissues of the firefly are extracted with water a solu tion which still gives light on filtration through filter paper is obtained. The light lasts a certain time and then disappears.
If the dried powdered luminous tissues of the firefly are allowed to come in contact with oxygen free water, no light is produced, but if we admit oxy gen, in a few minutes we find the mixture becomes luminous. If we wait for an hour or more before admitting oxygen, no light is produced. The appa ratus shown in figure 1 is convenient for demon strating this.
The material to be extracted is placed in the vessel C (fig. 1), provided with a ground-in stopper connected with a 120° stopcock. The water to be rendered free of oxygen is placed in B after passing hydrogen through stopcock C and closing it. B is connected through A with a hydrogen generator. The hydrogen is passed through potassium hydroxide to remove acid and then over a glowing platinum wire (in A) to remove the last traces of oxygen, a much better method than passing the gas through alkaline pyrogallol. By alternately exhausting B through b, connected to an air-pump, and refilling with hydrogen several times, the water can be quickly rendered free of oxygen. C is then connected to B through c and one of the arms of the 120° stop cock (d) whose other arm is connected with an air-pump. C and the arms of d are then exhausted. The 120° stopcock is then turned to connect C and B and c is opened, allowing the pressure of the hydrogen to drive the solvent on the material in C. The proper amount of fluid for extraction should be placed in B, so that the hydrogen may follow it through and fill the chamber C. Then d is closed, when C can be disconnected and shaken during extraction. To filter the extract it is only necessary to connect one of the arms of d with a desiccator fit ted with a funnel and filter rack. When the desiccator is exhausted, C and the desiccator are connected, and the pressure of the hydrogen in C drives the extract onto the filter paper. The firefly photogen begins to phosphoresce when the atmospheric pressure reaches 5 to 6 mm., which means an oxygen pressure of 1 to 1.2 mm. Consequently it is necessary to use a good vacuum-pump and make connections air-tight. I found small-bore lead tubing sealed with Khotinsky cement the best for the purpose.
All of my experiments have been carried out in the dark and the material observed at critical stages (as when the oxygen-free water was added) to make sure that no light appeared, and always with negative results. But to make sure that no very slow leakage of oxygen into the filtering chamber occurred, I have carried out the extraction in a special tube provided with a capillary sealed off during the extraction. After extracting in this tube for 14 hours and admitting oxygen, no phosphorescence appeared. Thinking that possibly the photogen dis
solved in the extracting fluid did phosphoresce, but only so faintly as to be invisible because distributed through a relatively large volume of extract fluid, the unfiltered extract was evaporated in vacuo to a small volume. This can be very easily done by placing the rubber tube from the vacuum-pump over the capillary onto the special tube, exhausting, and then breaking the capillary through the walls of the rubber tube to connect with the air-pump. Even when concentrated the extract gave no light on adding oxygen.
We must conclude that the photogen is destroyed in distilled water, even without oxidation. The search for watery solvent for the photo gen becomes, then, a search for a solvent in which the photogen is stable. The following solutions were tried in addition to distilled water. Extraction was allowed to proceed for from 1 to 14 hours.
In each case, after extraction, oxygen was admitted and the solution shaken, yet in no case did light appear either in the undissolved residue or in the solution. The 0.02 m HC1 extract was also neutralized, as it is well known that the acid prevents light-production. As McDermott (z9) has simultaneously failed to extract a photogenic substance with oxygen-free aqueous solvents, we may safely conclude that something connected with light-production undergoes decomposition on standing. We shall see later what this substance is.
Like Cypridina photogenin, firefly photogenin is prepared by allow ing an aqueous extract of the luminous organ to stand until the light disappears, and the photophelein is prepared by extracting the firefly with boiling water. Light appears on mixing the two substances. As we shall see, firefly photophelein alone in solution is very stabile, but, together with unboiled extract of luminous or non-luminous parts of the firefly, it is very unstabile. Therefore the photophelein is prob ably the substance which underwent decomposition when dried pow dered luminous tissue was allowed to stand in contact with oxygen-free water for one hour in the experiments described above.