A very small amount of luciferase only is necessary because it behaves as an enzyme and follows the general rule that catalysts act in minute concentrations.
On the assumption that luciferase is an enzyme, an organic catalyst oxidizing luciferin with light production, we may appropriately inquire into the relation between the concentration of luciferin and luciferase and intensity and duration of luminescence. Oxygen tension, hydrogen ion concentration and temperature must be maintained constant as these all affect both intensity and duration of luminescence. Before considering luciferin and luciferase, however, let us study a few well-known chemiluminescent oxidations with special reference to concentration of reacting substances and temperature.
The effect of temperature on luminescence is of special interest because it gives us a means of analysis for determining if the luminescence depends on reaction velocity. We know that photochemical reactions are very little affected by temperature because the reaction is dependent on the absorption of light, a physical process, and this increases only a small per cent. for a rise of temperature of 10° C. To put it in the usual way, its temperature coefficient (Q10) for a 10° interval is usually less than 1.1. On the other hand, we should expect photogenic reactions, in which some of the chemical energy is converted into radiant energy, to give off much more light the greater the reaction velocity. As reaction velocity increases so rapidly with temperature (Q10 = 2 to 3), luminescence intensity should rapidly increase with increase in temperature.
Trautz (1905), from his extensive study of the chemiluminescence of phenol and aldehyde compounds came to the conclusion that luminescence intensity was proportional to reaction velocity. He based his conclusions largely on the effects of temperature and concentration of reacting substances and went so far as to declare that any reaction would produce luminescence if the reaction velocity were sufficiently increased. It is quite true that increasing the temperature does increase the intensity of chemiluminescence, but this is only within certain limits. As we raise the temperature, chemiluminescence becomes more intense but we soon reach a temperature for maximum luminescence and above this the intensity diminishes. This is especially well seen in the action of various oxidizers on pyrogallol and H2O2 recorded in [Table 10]. At 100° C. practically no light is produced by many
oxidizers which are themselves unaffected at 100°. If we are to connect reaction velocity with intensity of luminescence we must conclude that the evolution of light is dependent rather on an optimum than a maximum reaction velocity.
TABLE 10
Temperature and Light Production. The Oxidizer is Mixed with an Equal Amount of M/100 Pyrogallol + 3 per cent. H2O2
| Oxidizer | Temperatures | ||||
|---|---|---|---|---|---|
| 0-2° | 20° | 50° | 75° | 98-100° | |
| Turnip juice | Faint | Good | Good | Bright | Negative. |
| 1 per cent. blood extract | Faint | Fair | Good | Fair. | |
| m/20 K4Fe(CN)6 | Negative | Good | Bright | Good. | |
| m/100 KMnO4 | Fair | Good | Bright | Bright | Faint flash. |
| m/50 K2Cr2O7 | Negative | Fair | Faint | Fair | Negative. |
| m/100 CrO3 | Negative | Good | Bright | Bright | Faint. |
| m/10 KCr alum | Negative | Faint | Faint | Faint | Negative. |
| m/10 NH4Fe alum | Negative | Faint | Faint | Faint | Very faint. |
| MnO2 | Negative | Fair | Fair | Fair | Negative. |
| NaClO | Bright flash | Bright flash | Bright flash | Fair flash. | |
Quite a number of instances are known in which increasing the mass of reacting substances leads not to an increase but to an actual cessation of luminescence. This fact does not confirm the theory that reaction velocity is a determining factor in luminescence. The conditions for the luminescence of white phosphorus are most interesting and unusual. (See van't Hoff, 1895; Ewan, 1895; Centnerszwer, 1895; Russell,1903; Scharff, 1908.) Phosphorus will only begin to luminesce at a certain small pressure of oxygen. This "minimum luminescence pressure" of oxygen is very low, so low that earlier observers, failing to remove traces of oxygen, thought that luminescence might occur in absence of oxygen. Curiously enough there is also a "maximum luminescence pressure" of oxygen above which no luminescence occurs. Phosphorus will not luminesce in pure oxygen. Between the minimum and maximum is an "optimum luminescence pressure" where luminescence of the phosphorus is brightest. The exact values of these pressures vary with degree of water vapor present and with temperature. According to Abegg's Handbuch der anorganischen Chemie, the maximum luminescence pressure with water vapor present, is 320 mm. Hg at 0° and increases 13.19 mm. Hg for each degree rise in temperature. This means that for a definite temperature, say, 20°, phosphorus will not luminesce with an oxygen pressure of 583 mm. Hg, but will luminesce with pressures under this. If, however, we raise the tempera