Table 13 — Substances Giving Light With Pyrogallol and Hydrogen Peroxide
| Equal volume added to mixture of 1 part M/100 pyrogallol or 1 part 3 per cent H2O2 + 1 part M/100 pyrogallol; hence, concentrations final mixture are one-half that given | Light with pyrogallol | Light with pyrogallol + H2O2 | Blueing of gum guaiac | Blueing of gum guaiac + H2O2 | Liberation of oxygen from H2O2 | ||
|---|---|---|---|---|---|---|---|
| 1 | Potassium ferrocyanide | (K4Fe(CN)6 M/10-M/20) | - | Bright | + | + | |
| 2 | Potassium ferricyanide | (K3Fe(CN)6 M/10-M/1,250) | - | Very faint to - | - | - | Very slow |
| 3 | Potassium chromate | (K2CrO4 M/20-M/100) | - | Good | + | + | |
| 4 | Potassium bichromate | (K2Cr2O7 M/50-M/100) | - | Good | + | + | |
| 5 | Potassium permanganate | (KMnO4 M/50-M/200) | - | Bright | + | - | + |
| 6 | Potassium hydroxide | (KOH M-M/6,250) | - | - | - | - | Very slow |
| 7 | Potassium chlorate | (KClO3 M/10) | - | - | - | - | - |
| 8 | Potassium persulfate | (K2S2O8 M/10-M/128) | - | - | - | - | - |
| 9 | Potassium chromium alum | (Cr2(SO4)3.K2SO4 M/10) | - | Faint | Very slow | Very slow | - |
| 10 | Ferric ammonium alum | (Fe2(SO4)3.(NH4)2SO4 M/10) | - | Faint | + | Very slow | |
| 11 | Ferric chloride | (FeCl3 M/10-M/250) | - | Fair | + | Slow | |
| 12 | Ferrous sulfate | (FeSO4 M/10-M/6,250) | - | Fair | - | + | Slow |
| 13 | Copper sulfate | (CuSO4 M/5-M/125) | - | - | - | + | Very slow |
| 14 | Chromic acid | (CrO3 M/100) | - | Bright | + | + | |
| 15 | Chromic sulfate | (Cr2(SO4)3 2 per cent) | - | Faint | - | + | Slow |
| 16 | Chlorine water | - | - | + | + | ||
| 17 | Bromine water | - | - | + | + | ||
| 18 | Iodine in KI | - | - | + | + | ||
| 19 | Sodium hypochlorite | (Cl water + NaOH) | Faint flash Bright | + | ++ | ||
| 20 | Sodium hypobromite | (NaOBr, bromine water + NaOH) | Faint flash Bright | + | ++ | ||
| 21 | Sodium hypoiodite | (I in KI + NaOH) | - | Faint | + | + | |
| 22 | Calcium hypochlorite | (Ca(OCl)2 saturated solution) | - | Good | + | ++ | |
| 23 | Turnip juice | - | Bright | - | + | ++ | |
| 24 | Turnip juice heated to 70° | - | Faint | - | + | Very slow | |
| 25 | Turnip juice boiled | - | - | - | - | - | |
| 26 | Albumin solution | - | - | - | - | - | |
| 27 | Albumin solution + KMnO4 | - | Good | + | - | ++ | |
| 28 | Albumin solution + KMnO4 boiled 1 min. and filtered (no precipitate forms) | - | Good | + | - | ++ | |
| 29 | Gelatin solution | - | - | - | - | - | |
| 30 | Gelatin solution + KMnO4 | - | Good | - | - | ++ | |
| 31 | Gelatin solution + KMnO4 boiled 1 min. and filtered (no precipitate forms) | - | Good | + | - | ++ | |
| 32 | Colloidal Ag | - | Bright | + | + | ||
| 33 | Colloidal Pt | - | Bright | + | + | ||
| 34 | Colloidal Fe(OH)2 (dilute) | - | - | - | + | - | |
| 35 | Sodium nucleoproteinate (liver) | - | - | - | + | - | |
| 36 | Sodium nucleoproteinate (mammary gland) | - | - | - | - | - | |
| 37 | Sodium nucleate (yeast) | - | - | - | - | - | |
| 38 | Squid blood (Sepia esculenta). Contains hemocyanin | - | Fair | ++ | |||
| 39 | Squid blood (Sepia esculenta) boiled | - | Good | - | |||
| 40 | Lobster blood (Palinurus japonicus). Contains hemocyanin and tetronerythrin, a lipochrome | - | Faint | ++ | |||
| 41 | Lobster blood (Palinurus japonicus) boiled | - | Fair | - | |||
| 42 | Annelid blood (Laonome japonica). Contains chlorocruorin | - | Good | ||||
| 43 | Annelid blood (Laonome japonica) boiled | - | - | ||||
| 44 | Luminous pennatulid extract (Cavernularia haberi) | - | - | - | + | ++ | |
| 45 | Luminous ostracod extract (Cypridina hilgendorfii) | - | - | + | |||
| 46 | Luminous protozoan extract (Noctiluca miliaris) | - | - | - | - | - | |
| 47 | Firefly (Luciola viticollis) extract, luminous organs | - | - | ++ | |||
| 48 | Ferrous ferrocyanide (Fe2Fe(CN)6) | - | Faint | + | + | ||
| 49 | Zinc ferrocyanide (Zn2Fe(CN)6) | - | - | + | Very slow | ||
| 50 | Chromic oxide (Cr2O3) | - | - | - | Slow | ||
| 51 | Chromic hydroxide (Cr(OH)2) | - | - | - | Slow | + | |
| 52 | Manganese dioxide (MnO2) | - | Good | Slow | Slow | ++ | |
I believe the explanation of these phenomena lies rather in another direction and that the effect of the temperature and concentration of reacting substances affects not only the reaction velocity but also the reaction products. While intensity of luminescence undoubtedly increases with increasing reaction velocity, the luminescence itself probably accompanies only one stage in the formation of a series of oxidation products. This stage is favored at a definite temperature and mass of reacting substances. Thus, in the oxidation of phosphorus several intermediate oxides are said to be formed. The oxidation takes place in steps and probably the luminescence is connected with only one of the steps in a chain of reactions. It is probable that a certain oxygen pressure and temperature favors that particular step at the expense of the others and so this oxygen concentration and temperature correspond to the optimum for luminescence.
The supposition that certain definite oxidation products of pyrogallol must be formed in order to produce light is borne out by the fact that pyrogallol must be oxidized in a particular way to obtain luminescence. The blackening of pyrogallol with absorption of oxygen in presence of alkali is a very well-known reaction, but luminescence does not accompany this type of oxidation. I have tried mixing all concentrations of pyrogallol and all concentrations of alkali in an endeavor to obtain some light, but always with negative results. Likewise my attempts to obtain light during the electrolysis of salt solutions containing pyrogallol by means of the nascent oxygen at various kinds of anodes have met with negative results. A similar case is presented by luciferin which oxidizes spontaneously (most rapidly in presence of alkali) without light production and only produces light when oxidized in presence of luciferase.
To sum up the results of the dynamics of chemiluminescence we may say that certain oxyluminescences occur only if the substance is oxidized in a particular way under definite conditions of temperature and concentration and that this is probably due to a favoring of one step (with which the luminescence is associated) in a chain of oxidations. Providing temperature and concentration are such as to favor the step responsible for luminescence, then higher temperature and greater concentration result in increased intensity of luminescence.
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 factors, such as time of heating, salt content, acidity, etc., so the luciferases of different animals coagulate at different temperatures depending on these conditions. Some of the more reliable observations on these critical temperatures are collected in Table 14.