The Nature of Animal Light Part 11

One of the most extraordinary things regarding luminescence in general is the small amount of material necessary to cause a visible emission of light. To take an extreme case, the flash of light resulting from the impact on ZnS of a single a particle, a helium atom, is visible to the naked eye. Addition of one part in a million of some heavy metal to pure CaS will confer phosph.o.r.escent properties on the latter. We are forced to believe that the heavy metal enters into some reaction during illumination which is reversed with light emission after illumination and a very small amount of heavy metal is necessary. Pyrogallol in water, 1:5,000,000 (m/512,000), can be oxidized with light production by K_{4}Fe(CN)_{6} and H_{2}O_{2} (Harvey, 1917) and m/100 pyrogallol + H_{2}O_{2} will give a visible light with colloidal platinum in 1:250,000 concentration (Goss, 1917).

Luciferin and luciferase from _Cypridina_ will also luminesce in exceedingly small concentration. If one grinds a single _Cypridina_ in a mortar with water and dilutes the extract to 25,600 c.c., light can be observed if luciferin is added to this dilute luciferase solution. By determining the volume of the luminous gland of _Cypridina_ and even a.s.suming that this volume is all luciferase, one can calculate that one part of luciferase in 1,700,000,000 parts of water will give light when luciferin is added. Likewise, a similar dilution of luciferin will give visible light when luciferase is added.

The sensitivity of our eye is largely responsible for the detection of so small an energy change. As we have seen, recent determinations have proved that the dark adapted eye can detect 18 10^{-10} ergs per second. From the heat of complete oxidation of pyrogallol it is possible to calculate the amount of pyrogallol necessary to give 18 10^{-10} ergs if completely oxidized. This quant.i.ty is infinitesimally small.

When pyrogallol is oxidized by K_{4}Fe(CN)_{6} and H_{2}O_{2}, it is not completely oxidized and probably only a small amount of the energy is converted into light; otherwise we should be able to see the luminescence of a very much weaker concentration of pyrogallol. As the reaction luciferin ? oxyluciferin is so easily reversible, very little energy must be liberated, and, as experiments indicate, very little heat, if any, accompanies light production. Even though this be true, it is still possible for a very small amount of luciferin to produce a very large amount of light.

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 a.s.sumption 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 a.n.a.lysis 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 (Q_{10}) 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 (Q_{10} = 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 H_{2}O_{2} 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. H_{2}O_{2}_

========================================================================= Temperatures Oxidizer +----------+---------+---------+--------+---------- 0-2 20 50 75 98-100 ----------------------+----------+---------+---------+--------+---------- Turnip juice Faint Good Good Negative.

1 per cent. blood extract Faint Fair Good Fair.

M/20 K_{4}Fe(CN)_{6} Negative Good Bright Bright Good.

M/100 KMnO_{4} Fair Good Bright Bright Faint flash.

M/50 K_{2}Cr_{2}O_{7} Negative Fair Faint Fair Negative.

M/100 CrO_{3} Negative Good Bright Bright Faint.

M/10 KCr alum Negative Faint Faint Faint Negative.

M/10 NH_{4}Fe alum Negative Faint Faint Faint Very faint.

MnO_{2} Negative Fair Fair Fair Negative.

NaClO Bright Bright Bright Fair flash flash flash flash.

Quite a number of instances are known in which increasing the ma.s.s 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 temperature, luminescence will occur with an oxygen pressure of 583 mm. Hg.

A somewhat a.n.a.logous case is presented by the oxidation of pyrogallol solution in contact with ozone, except that in this reaction too high a concentration of pyrogallol will hinder the oxidation. I have not studied the effect of varying concentrations of ozone. If oxygen, pa.s.sed through an ozonizer (the silent electric discharge tube), is bubbled through m/100 pyrogallol, no luminescence occurs at 0, a fair luminescence at 20, a good luminescence at 50, and a bright luminescence at the boiling point. If the pyrogallol is of _m_ concentration, no luminescence occurs at 0 or 20, a fair luminescence at 50, and a bright luminescence at the boiling point. For a definite temperature, say 20, no light appears if the pyrogallol is of _m_ concentration, but if we raise the temperature, luminescence can occur.

The similarity to phosphorus is obvious. Thus the "maximum luminescence pressure" of pyrogallol increases with increase of temperature.

We have already seen that pyrogallol can also be oxidized, if H_{2}O_{2} is present, by a great variety of substances, such as peroxidases of potato or turnip juice, haemoglobin, KMnO_{4}, K_{4}Fe(CN)_{6}, CrO_{3}, MnO_{2}, hypochlorites and hypobromites, or colloidal Pt and Ag. For convenience we may collectively speak of these as oxidizers. They are recorded in Table 13. No light occurs if H_{2}O_{2}is absent. In the case of some of these oxidizers pyrogallol will luminesce in dilute concentrations but not in strong. Also, dilute pyrogallol will luminesce with a dilute solution of oxidizer but not with a concentrated solution of oxidizer. The effect of rise in temperature in these cases also is to increase the "maximum luminescence concentration" of pyrogallol and the "maximum luminescence concentration" of oxidizer. Table 11 shows this effect of temperature with K_{4}Fe(CN)_{6} and varying concentrations of pyrogallol, and Table 12 shows the effect of temperature with pyrogallol and varying concentrations of K_{4}Fe(CN)_{6}. Table 10 shows the relation between temperature and intensity of luminescence with pyrogallol and various oxidizers. The terms _faint_, _fair_, _good_, and _bright_ are purely relative designations of brightness as estimated by the eye, for accurate measurements of weak intensities are very difficult to make.

From Table 10 it should be noted that the intensity of luminescence of pyrogallol oxidized with most oxidizers is actually less at the boiling point, a fact which I have repeatedly verified. Let us now see how these facts are to be explained. If we a.s.sume that luminescence is dependent on reaction velocity, the intensity of luminescence should increase with increasing temperature. Up to a certain limit this is what we find, but at temperatures above this limit the intensity of luminescence actually decreases. The duration of luminescence also decreases. There is an optimum temperature for luminescence in many cases and we can only conclude that luminescence depends not on a very rapid reaction velocity but on a certain definite reaction velocity. a.s.suming that this is true, how can we account for the anomalous fact that in high concentrations of oxygen, phosphorus will not luminesce or that in high concentrations of pyrogallol, there is no luminescence in presence of ozone or of oxidizer and H_{2}O_{2}. Of course with high active ma.s.s of oxygen (in case of phosphorous luminescence) or of pyrogallol (in case of pyrogallol luminescence) the reaction velocity must be greater than the optimum. If that is the case, then lowering the temperature should reduce the reaction velocity to the optimum and light should appear.

However, as we have seen, not lowering but raising the temperature causes luminescence with high oxygen concentration or high pyrogallol concentration.

TABLE 11

_Temperature, Concentration of Pyrogallol, and Light Production. An Equal Amount of M/20 K_{4}Fe(CN)_{6} is Mixed with Pyrogallol + 3 per Cent H_{2}O_{2}_

A = Concentration of pyrogallol (after mixing)

========================================================================= Temperatures A +---------+---------+-------+--------+--------+--------+--------- 0-2 10 20 30 50 75 98-100 --------+---------+---------+-------+--------+--------+--------+--------- M/4 Negative Negative Good Very Faint Fair Faint faint M/40 Negative Faint Faint Faint Good Bright Good M/400 Faint Fair Good Good Good Bright Bright flash M/4,000 Bright Bright Bright Bright Bright Fair Negative flash flash --------+---------+---------+-------+--------+--------+--------+---------

TABLE 12

_Temperature, Concentration of Ferrocyanide and Light Production. An Equal Amount of K_{4}Fe(CN)_{6} is Mixed with M/100 Pyrogallol + 3 Per Cent H_{2}O_{2}_

A = Concentration of K_{4}Fe(CN)_{6} exposed to light (after mixing)

======================================================================== Temperatures A +---------+-----+-----+-----+------+--------+------- 0-2 10 20 30 50 75 98-100 --------------------+---------+-----+-----+-----+------+--------+------- Half saturated Negative Faint Fair Fair Good Good Faint at 20 C flash One-sixth saturated Very Fair Good Good Bright Very Good at 20 C faint bright flash --------------------+---------+-----+-----+-----+------+--------+-------

TABLE 13

_Substances Giving Light with Pyrogallol and Hydrogen Peroxide_

Key to column headings:

A = Equal volume added to mixture of 1 part M/100 pyrogallol or 1 part 3 per cent H_{2}O_{2} + 1 part M/100 pyrogallol; hence, concentrations final mixture are one-half that given

B = Light with pyrogallol

C = Light with pyrogallol + H_{2}O_{2}

D = Blueing of gum guaiac

E = Blueing of gum guaiac + H_{2}O_{2}

F = Liberation of oxygen from H_{2}O_{2}

============================================================================= A B C D E F ---------------------------------------------+------+------+-----+-----+----- 1 Pota.s.sium ferrocyanide - Bright + + (K_{4}Fe(CN)_{6} M/10-M/20) 2 Pota.s.sium ferricyanide - Very - - Very (K_{3}Fe(CN)_{6} M/10-M/1,250) faint slow to - 3 Pota.s.sium chromate - Good + + (K_{2}CrO_{4} M/20-M/100) 4 Pota.s.sium bichromate - Good + + (K_{2}Cr_{2}O_{7} M/50-M/100) 5 Pota.s.sium permanganate - Bright + - + (KMnO_{4} M/50-M/200) 6 Pota.s.sium hydroxide - - - - Very (KOH M-M/6,250) slow 7 Pota.s.sium chlorate - - - - - (KClO_{3} M/10) 8 Pota.s.sium persulfate - - - - - (K_{2}S_{2}O_{8} M/10-M/128) 9 Pota.s.sium chromium alum - Faint Very Very - (Cr_{2}(SO_{4})_{3}.K_{2}SO_{4} M/10) slow slow 10 Ferric ammonium alum - Faint + Very (Fe_{2}(SO_{4})_{3}.(NH_{4})_{2}SO_{4} slow M/10) 11 Ferric chloride - Fair + Slow (FeCl_{3} M/10-M/250) 12 Ferrous sulfate - Fair - + Slow (FeSO_{4} M/10-M/6,250) 13 Copper sulfate - - - + Very (CuSO_{4} M/5-M/125) slow 14 Chromic acid - Bright + + (CrO_{3} M/100) 15 Chromic sulfate - Faint - + Slow (Cr_{2}(SO_{4})_{3} 2 per cent) 16 Chlorine water - - + + 17 Bromine water - - + + 18 Iodine in KI - - + + 19 Sodium hypochlorite Faint Bright + ++ (Cl water + NaOH) flash 20 Sodium hypobromite Faint Bright + ++ (NaOBr, bromine water + NaOH) flash 21 Sodium hypoiodite - Faint + + (I in KI + NaOH) 22 Calcium hypochlorite - Good + ++ (Ca(OCl)_{2} saturated solution) 23 Turnip juice - Bright - + ++ 24 Turnip juice heated to 70 - Faint - + Very slow 25 Turnip juice boiled - - - - - 26 Alb.u.min solution - - - - - 27 Alb.u.min solution + KMnO_{4} - Good + - ++ 28 Alb.u.min solution + KMnO_{4} boiled 1 min. and filtered (no precipitate forms) - Good + - ++ 29 Gelatin solution - - - - - 30 Gelatin solution + KMnO_{4} - Good - - ++ 31 Gelatin solution + KMnO_{4} 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 j.a.ponicus). Contains hemocyanin and tetronerythrin, a lipochrome - Faint ++ 41 Lobster blood (Palinurus j.a.ponicus) boiled - Fair - 42 Annelid blood (Laonome j.a.ponica). Contains chlorocruorin - Good 43 Annelid blood (Laonome j.a.ponica) 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 (Fe_{2}Fe(CN)_{6}) - Faint + + 49 Zinc ferrocyanide (Zn_{2}Fe(CN)_{6}) - - + Very slow 50 Chromic oxide (Cr_{2}O_{3}) - - - Slow 51 Chromic hydroxide (Cr(OH)_{2}) - - - Slow + 52 Manganese dioxide (MnO_{2}) - 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 ma.s.s 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 a.s.sociated) 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 haemoglobin saturation--oxygen tension curve. Haemoglobin 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 a.s.suming 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.