Let us now turn to luminous organisms and consider the effect of temperature and of concentration of reacting substances (oxygen, luciferin and luciferase) on the luminescence. We have already seen that luminescence of a luciferin-luciferase mixture begins with an extraordinarily low oxygen tension and increases in intensity with increasing tension of oxygen, but that very soon an oxygen tension is reached where a maximum luminescence is obtained and further increase of oxygen tension gives no brighter light. In this respect the luminescence intensity—oxygen tension curve is no doubt very similar to the hæmoglobin saturation—oxygen tension curve. Hæmoglobin is about 50 per cent. saturated at 10 mm. oxygen pressure, 80 per cent. saturated at 20 mm. oxygen pressure
and completely saturated at pressures of oxygen well below the pressure of oxygen in air (152 mm. Hg). As the optimum oxygen tension for luminescence of luciferin is also well below that of air, mixtures of luciferin and luciferase luminesce with equal brilliancy whether air or pure oxygen is bubbled through them. To obtain an excess of oxygen it is only necessary to keep the solution saturated with air and statements regarding concentration of luciferin and luciferase and intensity or duration refer to excess of oxygen. Investigators who have studied the effect of increase in oxygen pressure on luminous animals have come to the same conclusions. High pressures of air or oxygen do not increase the intensity of luminescence (Dubois and Regnard, 1884).
The hydrogen ion concentration of crude solutions of luciferin and luciferase, made by extracting whole Cypridinas with hot or cold water is fairly constant, about Ph = 9, determined electrometrically. Such solutions have a high buffer value and the Ph does not change during oxidation of luciferin so that this variable is automatically controlled.
Because of difficulties in measuring low intensities of light which are constantly changing, no figures on light intensities can be given, but it is easy to establish the following facts: The greater the concentration of luciferin or luciferase the more intense the luminescence. The greater the concentration of luciferin the longer the duration of luminescence and the greater the concentration of luciferase, the shorter the luminescence lasts. Thus, if we mix concentrated luciferin and weak luciferase we get a bright light which lasts for a half hour or more, gradually growing more dim. Concentrated luciferase and weak
luciferin give a bright flash of light which disappears almost instantly. Concentrated luciferase and concentrated luciferin give a brilliant light which lasts for an intermediate length of time and weak luciferin and weak luciferase give a faint luminescence which lasts for an intermediate length of time.
These facts can all be explained by regarding luciferase as a catalyzer which accelerates the oxidation of luciferin and by assuming that intensity of luminescence is dependent on reaction velocity, i.e., on rate of oxidation. Contrary to the condition for phosphorus and for pyrogallol there appears to be no optimum concentration of luciferase or luciferin, but the luminescence intensity increases gradually with increasing concentration of luminous substances up to the point where pure (?) luciferin and pure (?) luciferase, as secreted from the gland cells of the animal, come in contact with each other. This, the maximum brightness, is not to be compared with the light of an incandescent solid, but is nevertheless visible in a well-lighted room, out of direct sunlight.
The effect of temperature on Cypridina luminescence also bears out the preceding conclusions. For a given mixture of luciferin and luciferase the light becomes more intense with increasing temperature up to a definite optimum and then diminishes in intensity. The diminution in intensity above the optimum is due to a reversible change in the luciferase so that its active mass diminishes. This change becomes irreversible in the neighborhood of 70° (depending on various conditions), where coagulation of luciferase occurs. Light will appear at 0° but it is far less intense than light at higher temperatures and it is more yellow in color. The light of optimum temperatures
is quite blue. The weaker light at temperatures above the optimum is also more yellow in color. I believe this difference in color is a function of the slowed reaction velocity, for a mixture of luciferin and luciferase which gives a bluish luminescence at room temperature, will give a weaker and yellowish luminescence if diluted with water. Dilution with water will slow the reaction velocity. If the difference in color were not real but due to change in color sensitivity of the eye with different intensities of such relatively weak light (Purkinje phenomenon), the weaker light should appear more blue. As the weaker light appears more yellow, I therefore believe the color difference is actual and not subjective.
A minimum, optimum, and maximum temperature for luminescence is observed in all luminous organisms. The minimum is usually very low. Luminous bacteria will still light at -11.5° C. The power to luminesce under ordinary conditions is not destroyed by exposure to liquid air, for, on raising the temperature, light again appears (Macfayden, 1900, 1902). Almost all organisms will luminesce at 0° C., and the luminescence minimum probably represents the point at which complete freezing of the luminous solution occurs. It is very low with bacteria because they are solutions in capillary spaces of very small size, a condition tending to lower the freezing point.
The luminescence maximum represents the point at which luciferase is reversibly changed so as to be no longer active. If the temperature is again lowered the luciferase again becomes active and light reappears. Some degrees above this, and in all forms well below the boiling point, luciferase is coagulated and destroyed. As the coagulation point of proteins depends on many