When the dried powdered luminous materia_The Nature of Animal Light

over the surface of water, it goes into solution and leaves luminous

diffusion and convection trails plainly visible in the water. Many

luminous marine forms give off a phosphorescent slime when they are

handled, which adheres to the fingers. It is not surprising that this

luminous matter should have early received a name. In 1872, Phipson

called it _noctilucin_ and described some of its properties. He regarded

the luminous matter which can be scraped from dead fish (luminous

bacteria) and the mucous secretion of _Scolopendra electrica_ or the

luminous matter of the glowworm to be this material, noctilucin, which,

"in moist condition, takes up oxygen and gives off CO_{2} and when dry

appears like mucin." Phipson says that it forms an oily layer over the

seas in summer (he probably refers to masses of dinoflagellates), is

liquid at ordinary temperatures and less dense than water, smells a

little like caprylic acid, is insoluble in water but miscible with it,

insoluble in alcohol and ether, dissolves with decomposition in mineral

acids and alkalies and contains no phosphorus. We can see from this

description that the word "noctilucin" does not indicate a chemical

individual, but it is the earliest attempt to definitely designate the

luminous substance.

The idea of a definite substance oxidizing and causing the light has

been upheld by a number of investigators, and many years later Molisch

called this substance the _photogen_. He contrasts the "photogen theory"

with certain other views of light production, which may be spoken of as

"vital theories," notably those of Pflueger (1875), who looked upon

luminescence as a sign of intense respiration, and of Beijerinck (1915),

who regarded the light as an accompaniment of the formation of living

matter from peptone.

Fortunately biological science has advanced beyond the stage where a

living process can be explained by calling it a vital process, and we

must fall back upon the idea of a photogen oxidizing with light

production. Indeed, it is now possible to go much further than this and

describe the properties of the photogen, but we must not lose sight of

the fact that it was recognized very early in the history of

Bioluminescence, that water, oxygen, and a photogenic substance were

necessary for light production.

A very great advance in our knowledge of the chemistry of the problem

was made by Dubois in 1885. He showed that if one dips the luminous

organ of _Pyrophorus_ in hot water, the light disappears and will not

return again. Also if one grinds up a luminous organ the mass will glow

for some time but the light soon disappears. If one brings the

previously heated organ in contact with the unheated triturated organ it

will again give off light. Later, Dubois showed that the same experiment

could be performed with the luminous tissues of _Pholas dactylus_. A

hot-water extract of the luminous tissue, and a cold-water extract of

the luminous tissue, allowed to stand until the light disappears, will

again produce light if mixed together. Dubois (1887 _b_) advanced the

theory that in the hot-water extract there is a substance, luciferin,

not destroyed by heating, which oxidizes with light production in the

presence of an enzyme, luciferase, which is destroyed on heating. The

luciferase is present together with luciferin in the cold-water extract,

but the luciferin is soon oxidized and luciferase alone remains. Mixing

a solution of luciferin and luciferase always results in light

production until the luciferin is again oxidized. Similar substances

have been found by me in the American fireflies, _Photinus_ and

_Photuris_, the Japanese firefly, _Luciola_, and in the ostracod

crustacean, _Cypridina hilgendorfii_. Crozier[6] reports that they exist

also in _Ptychodera_, a balanoglossid. I have been unable to demonstrate

their existence in luminous bacteria; in the annelid, _Chaetopterus_; the

pennatulids, _Cavernularia_ and _Pennatula_; the squid, _Watasenia_;

and the fish, _Monocentris japonica_. E. B. Harvey (1917) could not

demonstrate them in _Noctiluca_. There are several reasons why the

existence of such bodies might be difficult to demonstrate, but these

reasons cannot be considered here. We thus see that the photogen is in

reality of dual nature, that two substances are necessary for light

production and that they may be very readily separated because of

difference in resistance to heating. In this respect Bioluminescence is

similar to some other biological processes, notably to certain immune

reactions and to certain enzyme actions.

[6] Private communication.

Thus, for the haemolysis of foreign red blood corpuscles, a specific

immune body (_amboceptor or substance sensibilatrice_) not destroyed by

moderate heating, and a thermolabile complement (_alexin_) are

necessary.

For the alcoholic fermentation of glucose by the zymase of yeast juice

two substances are also necessary. The zymase is made up of a heat

resistant, dialyzing component, the co-enzyme, and a non-dialyzing

substance, destroyed on boiling, the enzyme proper. Both must be present

for alcoholic fermentation of glucose to proceed and the two may be

separated by dialysis or by their difference in resistance to heating.

Several other characteristics of living cells are known to depend on the

joint action of two substances, one thermolabile, the other

thermostable. The reducing action of tissues, according to Bach,

requires a reducing enzyme proper or perhydridase and some easily

oxidizable substance, such as an aldehyde. The aldehyde has been spoken

of as the co-enzyme.

Because of the necessity of thermostable and thermolabile substances for

light production in luminous animals and because I was unable to oxidize

the thermostable material of _Cypridina_ with such oxidizing agents as

KMnO_{4}, H_{2}O_{2}, blood and H_{2}O_{2}, BaO_{2}, etc., I called the

heat resistant substance of _Cypridina_, "_photophelein_" (from _phos_,

light and _opheleo_, to assist), comparable to co-zymase, and the heat

sensitive substance of _Cypridina_, "photogenin" (from _phos_, light and

_gennao_, to produce), comparable to the zymase proper of yeast. In mode

of preparation and properties, the photophelein of _Cypridina_ was also

comparable to the luciferin of _Pholas_ and the _photogenin_ of

_Cypridina_ to the luciferase of _Pholas_. I also regarded photogenin as

the source of the light (hence the name), because a solution of

_Cypridina_ photogenin (=_Pholas_ luciferase) will give light on mixing

with crystals of salt and other substances which could not possibly be

oxidized. I later found, however, that this result was due to the fact

that the photogenin solution contained some of the thermostable

substance (luciferin) bound (combined or adsorbed), and that this was

freed by the salt crystals and oxidized with light production. I have

consequently abandoned the view that the system of substances concerned

in light production is similar to the zymase--co-zymase system of

yeast--and have adopted Dubois' term, luciferase (=_photogenin_) for the

thermolabile material, and luciferin (=_photophelein_) for the

thermostable material.

The luciferin of _Cypridina_ differs from that of _Pholas_ in that it

will not oxidize with light production with any oxidizing agents that I

have tried, and will give no light with luciferase from _Pholas_. It

does, however, oxidize spontaneously in solution, although no light

accompanies this oxidation.

I believe that for accuracy and definiteness we must designate the

luciferins and luciferases from different animals by prefixing the

generic name of the animal and speak of _Pholas_ luciferin, _Cypridina_

luciferase, _Pyrophorus_ luciferase, etc. In extracts of many

non-luminous animals Dubois has found oxidizing agents which can oxidize

_Pholas_ luciferin with light production and I have confirmed this for

_Pholas_, but I have not found any such substances in non-luminous

animals which will oxidize _Cypridina_ luciferin with light production.

I have found in extracts of non-luminous animals substances which will

liberate the bound luciferin in a concentrated _Cypridina_ luciferase

solution. The luciferin can then be oxidized by the luciferase and light

appears. Their effect is similar to that of salt crystals and I suggest

that they be called _photopheleins_, substances that assist in the

luciferin-luciferase reaction by liberating bound luciferin. One of the

best ways of freeing a solution of luciferase from bound luciferin is to

shake with chloroform. We can then do away with the disturbing effects

of bound luciferin.

It is obvious that luciferin must be formed from some precursor in the

cell and following the usual biochemical terminology, Dubois has called

it _proluciferin_ or _preluciferin_, and believes that it is converted

into luciferin by an enzyme co-luciferase. The experiments to prove the

existence of proluciferin were first made by Dubois on _Pholas_ in 1907

and have since been amplified (1917 _a_; 1918 _a_ and _b_).

In order to understand these experiments it must be borne in mind that

Dubois prepares luciferin from _Pholas_ in three ways: (1) By

precipitating the viscid luminous fluid from the siphons with 95 deg.

alcohol and dissolving the precipitate in water (1901_a_, 1907). (2) By

extracting the luminous organs with 90 deg. alcohol in a closed vessel for

twelve hours and filtering (1896). (3) By heating the viscid luminous

fluid to 70 deg. C. Apparently _Pholas_ luciferin is sparingly soluble in

alcohol as it can be obtained either in an alcoholic extract (method 2)

or by precipitation with alcohol (method 1). Proluciferin (called

_preluciferine_ in a later paper, 1917 _a_, 1918 _a_), is prepared by

methods 1 or 2 except that fatigued siphons, from which luciferin has

been removed by washing, are used (1907, 1917 _a_, 1918 _a_).

Preluciferin can also be obtained on boiling an extract of the luminous

organ of _Pholas_ because luciferin (at 70 deg.), luciferase (at 60 deg.) and a

co-luciferase are all destroyed below the boiling point (1917 _a_).

Co-luciferase is prepared (1) by heating a luciferase solution to 65 deg.

(1917 _a_) or (2) by extracting with water portions of the siphon of

_Pholas_ which have previously been macerated and well extracted with

alcohol (1918 _a_). Long-continued treatment with alcohol apparently

destroys the luciferase without affecting the co-luciferase. On mixing a

solution of preluciferin with one of co-luciferase and allowing them to

stand for 8-10 hours, luciferase is formed and can be recognized by the

fact that it will give light with a crystal of KMnO_{4}. Preluciferine

does not do this.

Recently Dubois (1918 _a_) states that preluciferine is nothing but

taurine and that taurine occurs in large quantities in _Pholas_ and is

transformed into luciferine by the action of co-luciferase. Not only

taurine, but also Byla's peptone, egg lecithin, and esculin can be

converted into luciferine by co-luciferase, and since esculin, a

glucoside, is so transformed, Dubois believes this proves that

co-luciferase belongs to the hydrolases. Indeed, it proves too much.

Luciferin must have an extraordinary chemical structure if it can be

formed by hydrolysis of such diverse compounds as peptone, lecithin,

esculin and taurine. A glance at the structural formula of esculin and

taurine is sufficient to emphasize the diverse nature of these two

substances.

[Illustration: Taurine]

[Illustration: Esculin]

I believe that in these experiments Dubois has been working with an

oxidation product of luciferin, what I have called _oxyluciferin_,

rather than a pro-substance. The mode of preparation of _Pholas_

preluciferin and _Pholas_ co-luciferase is such as could be used in the

preparation of _Cypridina_ oxyluciferin, and it seems more logical to

look for the presence of _Pholas_ oxyluciferin in one or both of Dubois'

extracts rather than believe that luciferin can be formed from both

taurine and esculin. When the co-luciferase solution stands with the

preluciferin solution we would in reality have not the formation of

luciferin from preluciferin, but the formation of luciferin from

oxyluciferin, by some reducing agent in the mixture. Indeed, in a very

recent paper Dubois (1919 _c_) takes the view that his co-luciferase is

a reducing enzyme which forms luciferin by reduction (presumably from

oxidized luciferin) and no mention is made of preluciferin.

It is, of course, obvious that when luciferin oxidizes, some oxidation

products must be formed. Most observers have assumed the oxidation

products of luciferin to be relatively simple and to represent a rather

complete breaking down of the luciferin molecule. Carbon dioxide was

mentioned by Phipson (1872) as being formed. We have just seen that no

carbon dioxide is formed during the oxidation of _Cypridina_ luciferin

and there is evidence that no fundamental change at all occurs. It is

for this reason that I have called the oxidation product of luciferin

_oxyluciferin_.[7] As we shall later see, the change luciferin

oxyluciferin is to be compared to the oxidation of colorless dyes

(leuco-compounds) to the colored dye. The chemical properties of

oxyluciferin are similar to those of luciferin and the oxyluciferin can

be readily reduced to luciferin again.

[7] It is unfortunate that Dubois (1918 b) has used the term

oxyluciferine in a quite different sense from the present use. He

regards oxyluciferine as a substance still capable of giving light by

autooxidation, and represents the steps in luminescence as follows:

"Co-luciferase + preluciferine = luciferine.

Luciferase + luciferine = oxyluciferine.

Oxyluciferine + oxygene = lumiere."

I should represent them as follows:

Luciferin + oxygen <=> oxyluciferin.

The reaction proceeds to right with light production only in presence of

luciferase.

Finally, we have the fluorescent substance of _Pyrophorus_ and

fireflies, which Dubois first called _pyrophorin_, but later, adopting

McDermott's terminology, speaks of as _luciferesceine_. This Dubois

regards as a substance intensifying the light and modifying its color by

changing invisible into visible rays. As we have seen, this theory,

while attractive, will not stand the test of critical examination.

Phipson's noctilucin, while the first name for the photogen of luminous

animals, is too vague a substance, chemically, to warrant a retention of

the term. Of the names, luciferin, luciferase, preluciferin or

proluciferin, co-luciferase, photogenin, photophelein, oxyluciferin,

luciferesceine, I believe that only proluciferin, luciferin,

oxyluciferin, luciferase and photophelein stand for substances which are

really significant for the theory of light production. _Luciferin_ is

the heat resistant, dialyzable substance which takes up oxygen and

oxidizes with light production in the presence of the heat sensitive,

non-dialyzing, enzyme-like _luciferase_. The luciferin must come from

some precursor, _proluciferin_, but I have been unable to demonstrate

the existence of this body in _Cypridina_ and know nothing definite of

its properties. The luciferin oxidizes to _oxyluciferin_ which has the

same chemical properties as the luciferin itself and may be reduced to

luciferin again by reducing substances in luminous and other animals or

by inorganic reducing agents. _Photophelein_ is a name for substances in

various animal or plant extracts which are capable of liberating

luciferin from some bound condition in solutions containing luciferase.

Under this term are included a number of unknown, probably quite

different substances, some of which are thermostable and others

thermolabile.

We have seen that Bioluminescence is an oxyluminescence, that the light

is probably due to the oxidation of a compound, luciferin, in presence

of air and water and that the oxidation is accelerated by an enzyme-like

substance, luciferase. We also saw in Chapter 2 that light production is

of fairly common occurrence during the oxidation of many organic

compounds, provided the oxidation is carried out in the proper way. Many

of these organic compounds must be oxidized by relatively strong alkali

or such strong oxidizing agents as would have a very deleterious action

on living cells. In 1913, Ville and Derrien, in a short note to the

French Academy, "Catalyse Biochemique d'une Oxydation Luminescente,"

show that _lophin_ could be oxidized by vertebrate blood in the presence

of H_{2}O_{2}. In the same year Dubois (1913) found that esculin, the

glucoside from horse chestnut bark, would also oxidize and luminesce in

presence of blood and H_{2}O_{2}. In these cases the haemoglobin of the

blood acts as a catalyst, transferring oxygen from the H_{2}O_{2} to

esculin or lophin and is to be compared to luciferase, except that

luciferase does not require the presence of H_{2}O_{2}.

As the haemoglobin does not lose this power on boiling, whereas

luciferase does, the analogy is far from perfect. Many oxygen carriers

are known, however, which may be destroyed on boiling their solutions,

namely, the peroxidases of plant juices. Esculin will not luminesce with

peroxidase and H_{2}O_{2}, but pyrogallol or gallic acid will. If one

mixes a test tube containing pyrogallol solution + H_{2}O_{2} with

potato or turnip juice or almost any plant extract, a yellowish

luminescence appears. The plant extract loses the power to cause such

luminescence on boiling and the peroxidase will not dialyze. It is, of

course, comparable to luciferase and acts on the thermostable,

dialyzable pyrogallol-H_{2}O_{2} mixture, which is comparable to

luciferin. Curiously enough, although many hydroxyphenol and

amino-phenol compounds can be oxidized by peroxidase and H_{2}O_{2},

only pyrogallol and gallic acid will oxidize with light production. Many

other oxidizers can take the place of the peroxidase. A list of these is

given on page 151. No other peroxide can take the place of H_{2}O_{2}

with peroxidases as oxidizers, but a few can replace H_{2}O_{2} with

other oxidizers. This is brought out in Table 7.

TABLE 7

_Peroxides Giving Light with Pyrogallol and Oxidizers_

Key to column headings:

[A]: Oxidizer. (Equal parts added to a mixture of M/100 pyrogallol and the

peroxide)

[B]: H_{2}O_{2} 3 per cent.

[C]: Benzoyl hydrogen peroxide (insoluble powder)

[D]: Ozonized turpentine (one drop)

[E]: Na_{2}O_{2} (powder)

[F]: BaO_{2} (powder)

[G]: MnO_{2} (insoluble powder)

[H]: PbO_{2} (insoluble powder)

[I]: K persulfate M/10

[J]: Na perborate M/20

[K]: K perchlorate M/10

[L]: Quinone (insoluble crystals)

-----------------------------------------------------------------------------

[A] |[B]|[C]|[D]| [E] | [F] |[G]|[H]| [I] | [J] |[K]|[L]

-------------------------+-- +---+---+-----+-----+---+---+-----+-----+---+---

Turnip juice | + | - | - | - | - | - | - | - | | | -

1 percent blood extract | + | - | - |Faint| - | - | - | - | - | - | -

M 20 K_{4}Fe(CN)_{6} | + | - | - | - | - | - | - | - | - | - | -

M 100 KMnO_{4} | + | - | - | - | - | - | - |Faint|Fair | - | -

M 10 FeCl_{3} | + | | | | | | | | - | - |

M 100 CrO_{3} | + | | | | | | | | - | - |

Na hypobromite | + | - | - |Faint|Faint| - | - |Fair |Fair | - | -

Ca hypochlorite | + | - | - | - | - | - | - |Faint|Fair | - | -

MnO_{2} | + | | | | | | | | | |

Mn(OH)_{3} sol in peptone| + | | | | | | | | - | - |

Colloidal Ag | + | | | | | | | | | |

-------------------------+---+---+---+-----+-----+---+---+-----+-----+---+---

Our knowledge of the existence of such analogous, purely organic

chemical oxidations, which proceed with light production, greatly

strengthens Dubois' theory that the luciferin-luciferase reaction

really represents a catalytic oxidation of similar nature. As Dubois

(1914 _a_) expresses it, we are dealing in luminous organisms with "1 deg.

une luminescence; 2 deg. une chemiluminescence; 3 deg. une oxyluminescence; 4 deg.

une zymoluminescence.

"Ou si l'on bien admettre que les zymases sont encore quelque chose de

vivant, une Biozymooxyluminescence." Perhaps it is not really necessary

to admit that the enzymes are living in order that we may adequately

visualize the nature of the photogenic process.

In the next chapter the properties of the three principal substances,

luciferin, oxyluciferin and luciferase, will be studied more carefully.

CHAPTER VI

THE CHEMISTRY OF LIGHT PRODUCTION, PART II

Since Radziszewski's experiments on the oxidation of oils in alcoholic

solutions of alkali, most of the early workers on Bioluminescence

tacitly assumed that the oxidizable material was fat or a fat-like

substance. Support was given to this view by the occurrence in cells of

granules or globules from which the light was seen to come. We now know

that these bodies are not fat droplets and that neither luciferin nor

luciferase are soluble in such fat solvents as ether, chloroform, benzol

or benzine. Phipson's description of the properties of noctilucin are

too crude and inaccurate to be considered. Dubois did not study the

chemical properties of luciferin and luciferase from _Pyrophorus_, the

first form with which he worked, except to point out that _Pyrophorus_

luciferase was destroyed on heating and was precipitated by alcohol

while the _Pyrophorus_ luciferin was not so affected. Luciferin was

found only in the luminous organ of _Pyrophorus_, not in the blood;

luciferase probably exists throughout the animal.[8]

[8] Private communication from R. Dubois.

PHOLAS LUCIFERIN.--In a series of papers since 1887 Dubois has studied

the chemical properties of _Pholas_ luciferin and _Pholas_ luciferase.

He finds the luciferin to be destroyed above 70 deg. C., to dialyze slowly,

to oxidize with light production in the presence of _Pholas_ luciferase,

KMnO_{4}, H_{2}O_{2}, haematine and H_{2}O_{2}, BaO_{2}, PbO_{2},

hypochlorites, and the blood of various marine mollusks and crustacea.

It is insoluble in fat solvents but forms a colloidal solution in water

from which it is precipitated unchanged by picric acid, alcohol at 82 deg.,

and (NH_{4})_{2}SO_{4}. It is not precipitated by NaCl, MgSO_{4} or

acetic and carbonic acids, except in presence of neutral salts. It forms

an insoluble alkali albumin with NH_{4}OH. Dubois (1887 _a_) stated at

one time that it could be crystallized and has spoken of it as belonging

to several different classes of substances, proteose, nucleoprotein,

albumin. Most recently he describes luciferin as a natural albumin

having acid properties. It occurs only in luminous, not in non-luminous

animals, and is found in all parts of the mantle, especially the

siphons. It does not occur in non-luminous parts of the mollusk. No

photographs of luciferin crystals have ever been published.

PHOLAS LUCIFERASE.--_Pholas_ luciferase has all the properties of an

enzyme, is destroyed at 60 deg. C., is non-dialyzable, insoluble in fat

solvents, but forms a colloidal solution in water. It is not affected by

1 per cent. NaF but its activity is suspended in saturated salt

solutions, sugar or glycerine, and it may be preserved in this way, its

activity returning on dilution. It is digested by trypsin and slowly

destroyed by the fat solvent anaesthetics, such as chloroform. For this

reason Dubois regards it as an oxidizing enzyme similar to the oxydones

of Batelli and Stern. Because he found iron in an extract of _Pholas_

dialyzed for a long time against running water, Dubois considers that it

is associated with iron, and reports that it will oxidize the ordinary

oxidase reagents, such as pyrogallol, gum guaiac, a-naphthol and

para-phenylene-diamine, etc. It remains to be proved, however, that

luciferase and not the oxidizing systems such as occur in all cells are

responsible for the coloration of these reagents. Dubois has found

luciferases or substances capable of giving light with _Pholas_

luciferin in the blood of many non-luminous crustacea and mollusks (in

_Barnea candida_, _Solen_, _Cardium edulis_, _Ostraea_ and _Mytilus_).

CYPRIDINA LUCIFERIN.--Despite the large amount that has been written on

luminous animals, Dubois' work on _Pholas_ and my own on _Cypridina_ and

the firefly are the only truly chemical studies that give us any idea of

the nature of the photogenic substances in any luminous animal. In many

ways _Cypridina_ luciferin is similar to _Pholas_ luciferin, but the two

differ in a sufficient number of points to make certain that they are

not identical substances. As I have emphasized above, we should speak

not of luciferin and luciferase but of the _luciferins_ and the

_luciferases_. The luciferins, as the oxidizable substances, must claim

first attention. They are more simple substances than the luciferases.

If we are to produce light artificially in the same way that animals do,

the luciferins must be synthesized. The luciferin of _Pholas_ will

luminesce with KMnO_{4} and other oxidizing agents, and, although I have

not yet succeeded in oxidizing _Cypridina_ luciferin with oxidizing

agents, I have no doubt but that some inorganic catalyzer will be found

to take the place of luciferase and accelerate oxidation of _Cypridina_

luciferin with light production.

The most remarkable peculiarity of _Cypridina_ luciferin is its

stability. In my first paper on _Cypridina_ I stated that luciferin was

not destroyed by momentary boiling but would be destroyed if boiled four

or five minutes; also, that it was unstable at room temperatures and

would disappear from solution in the course of a day or so. The reason

for this is that luciferin oxidizes even in absence of luciferase and

will then no longer give light with luciferase. This spontaneous

oxidation, which occurs without light production, can be prevented by

keeping the luciferin in a hydrogen atmosphere or by the addition of

acid. Under these conditions the luciferin can be boiled without

destruction or preserved for months without deterioration. The rapid

disappearance of luciferin from neutral or alkaline solution on boiling

in the air is entirely due to the more rapid oxidation at the boiling

point. As the oxidation product, oxyluciferin, can be readily

reconverted into luciferin again, we can not consider luciferin unstable

in the sense that its molecule is actually destroyed as is the case when

luciferase is boiled.

Not only is luciferin stable on boiling but it will actually withstand

boiling for 10 hours with 20 per cent. HCl (by weight, sp. gr. = 1.1) or

with 4 per cent. H_{2}SO_{4}. After one day of boiling with 20 per cent.

HCl the luciferin was completely destroyed and with 4 per cent.

H_{2}SO_{4} destruction was almost complete. In these cases there was no

question of a mere oxidation to oxyluciferin, as no oxyluciferin could

be demonstrated after boiling with such strong acids. An actual

destruction, probably an hydrolysis of the luciferin molecule, occurred.

We shall have occasion to refer to this again in considering the protein

nature of luciferin. It must be borne in mind that many proteins require

four or five days' boiling with 20 per cent. HCl for complete hydrolysis

to amino-acids. Luciferin forms a solution in water, probably colloidal,

although the luciferin will dialyze through parchment or collodion

membranes. It is rather readily adsorbed by various finely divided

materials such as bone black, Fe(OH)_{3}, kaolin, talc and CaCo_{3}. It

is not destroyed by any of the enzyme solutions which I have tried.

These include such as are widely divergent in action: pepsin HCl,

trypsin, erepsin, salivary and malt diastase, yeast invertase, urease,

rennin and the enzymes of dried spleen, kidney and liver substances.

By extracting the dried Cypridinas ground to a powder, the solubility of

luciferin in non-aqueous solvents could be easily studied, and by adding

such reagents as dilute acids, alkalies, neutral salts and the

alkaloidal reagents to an aqueous solution of luciferin the general

biochemical behavior of luciferin can be quite accurately stated. For

convenience the results of this study are given in Table 8.

TABLE 8

_Properties of Photogenic Substances from Cypridina_

=========================================================================

Property | Luciferase | Luciferin

------------------------+-----------------------+------------------------

_Salting out_ | |

By saturation NaCl |Not precipitated |Not precipitated.

By half saturation | Do. | Do.

MgSO_{4} | |

By saturation MgSO_{4}|Nearly completely |Partially

| precipitated | precipitated.

By saturation MgSO_{4}| ... | Do.

+ acetic acid | |

By half saturation | |

(NH_{4})_{2}SO_{4} |Slightly precipitated |Not precipitated.

By saturation |Completely precipitated|Nearly completely

(NH_{4})_{2}SO_{4} | | precipitated.

By saturation | |

(NH_{4})_{2}SO_{4} +| ... |Nearly completely

acetic acid | | precipitated.

| |

_Solubility in_ | |

Methyl alcohol |Insoluble |Soluble.

Ethyl alcohol | Do. | Do.

90 per cent. | Do. | Do.

70 per cent. | Do. | Do.

50 per cent. |Slightly soluble | Do.

Propyl alcohol |Insoluble | Do.

Isobutyl alcohol | Do. |Fairly soluble.

Amyl alcohol | Do. |Slightly soluble.

Benzyl alcohol | Do. |Soluble.

Acetone | Do. |Fairly soluble.

90 per cent. | Do. |Soluble.

70 per cent. |Slightly soluble | Do.

50 per cent. |Fairly soluble | Do.

Ethyl acetate |Insoluble | Do.

Ethyl propionate | Do. | Fairly soluble.

Ethyl butyrate | Do. | Do.

Ethyl valerate | Do. |Slightly soluble.

Ethyl nitrate | Do. |Very slightly soluble.

Glycerine | Do. |Soluble.

Glycol | Do. | Do.

Ether | Do. |Insoluble.

Chloroform | Do. | Do.

Carbon disulfide | Do. | Do.

Carbon tetrachloride | Do. | Do.

Benzol | Do. | Do.

Toluol | Do. | Do.

Xylol | Do. | Do.

Petroleum ether | Do. | Do.

Anilin | Do. | Do.

Glacial acetic acid | Do. |Fairly soluble.

| |

_Alkaloidal Reagents_ | |

Phosphotungstic acid |Completely precipitated|Very nearly completely

| | precipitated.

Phosphotungstic and | ... |Very nearly completely

acetic acids | | precipitated.

Phosphotungstic acid | ... |Completely precipitated.

and HCl | |

Tannic acid |Nearly completely |Nearly completely

| precipitated | precipitated.

Tannic and acetic | ... |Nearly completely

acids | | precipitated.

Tannic acid and HCl | ... |Nearly completely

| | precipitated.

Picric acid |Nearly completely |Not precipitated.

| precipitated |

Picric and acetic acid| ... | Do.

Picric acid and HCl | ... | Do.

K_{4}Fe(CN)_{6} and | ... |

acetic acid | | Do.

| |

_Heavy Metal Salts_ | |

Basic lead acetate |Completely precipitated|Not completely

| | precipitated.

Neutral lead acetate |Nearly completely |Not completely

| precipitated. | precipitated.

Neutral lead acetate | ... |Not precipitated.

and acetic acid | |

Mercuric chloride |Not precipitated |Not completely

| | precipitated.

Mercuric chloride and | ... |Almost completely

acetic acid | | precipitated.

Uranyl nitrate and | ... |Not completely

acetic acid | | precipitated.

| |

_Acids and Alkalies_ | |

NaOH |Not precipitated |Not precipitated.

NH_{4}OH | Do. | Do.

Acetic acid | Do. | Do.

H_{2}CO_{3} | Do. | Do.

Trichloracetic acid | Do. | Do.

------------------------+-----------------------+------------------------

Because the luciferin is almost completely precipitated by saturation

with (NH_{4})_{2}SO_{4}, we may conclude that it occurs in water in the

colloidal state. This excludes it from belonging to one of the numerous

groups of biochemical compounds occurring in true solution and places it

among the known groups of colloidal substances, the soaps, proteins,

polysaccharides, phospholipins, galactolipins (_cerebrosides_), tannins

or saponins. It is not a polysaccharide because nearly completely

precipitated by phosphotungstic acid, nor a soap because not

precipitated by calcium salts, nor a phospho- or galactolipin because

insoluble in benzine, hot or cold. It gives no tannin or saponin tests.

Only the protein group remains, and of the eighteen protein classes

recognized by the American Society of Biochemists, the general

properties of luciferin indicate that it should be placed among the

natural proteoses, somewhere on the borderland between the proteoses and

peptones. The fact that luciferin will dialyze, although almost

completely salted out by (NH_{4})_{2}SO_{4}, is strong evidence in favor

of placing it in such a position.

On the other hand, luciferin has two properties which to say the least

are unusual for proteins. I refer to its solubility in alcohols,

acetone, esters, etc., and non-digestibility by trypsin or erepsin,

which have almost universal proteolytic power.

The best known class of proteins soluble in alcohol is the prolamines of

plants, but the prolamines are insoluble in water and in absolute

alcohol. Zein, the prolamine of corn, is soluble in 90 per cent. ethyl,

methyl, and propyl alcohols, in glycerol heated to 150 deg. C., and in

glacial acetic acid. Recently Osborne and Wakeman (1918) have described

a protein from milk having solubilities similar to those of gliadin, the

prolamine of wheat. Welker (1912) has described a substance, obtained

from Witte's peptone, giving the biuret, Millon, and Hopkins-Cole tests,

which is soluble in water and absolute alcohol but not in ether, and it

is possible that others of the peptones are soluble in absolute alcohol.

On the other hand, some proteins in the absence of salts form colloidal

solutions in strong alcohol from which they may be precipitated by an

appropriate salt. As the absolute alcohol extract of _Cypridinae_ was

made from dry material containing the salts of sea water, some salt was

present, but there is always the possibility of sol formation.

If we extract dried _Cypridinae_, which have previously been thoroughly

extracted with benzine or ether, with 800 c.c. of boiling absolute

alcohol for an hour, filter the alcohol extract through blotting paper

and hardened filter paper, quickly evaporate the filtrate to dryness on

the water bath, and dissolve the residue in a small quantity of water

saturated with CO_{2},[9] we obtain a yellow opalescent solution which

gives a bright light with luciferase. This solution contains some

protein or protein derivatives as it gives a very faint Millon

reaction, a good positive ninhydrin test, reddish blue in color, but no

biuret reaction. It precipitates with tannic and phosphotungstic acids

but not with picric, acetic, trichloracetic, or chromic acids. The

extract gives a faint Molisch reaction for carbohydrates. As the

evidence points to the presence of some protein products in the absolute

alcohol extract of _Cypridinae_, it is possible that this protein is

luciferin. It should be emphasized, however, that the Millon reaction

was very faint, although the ninhydrin was quite marked and the biuret

negative.

[9] To make the solution slightly acid and prevent oxidation of the

luciferin.

Although luciferin is not digested by trypsin, even after five days at

38 deg. C., it does hydrolyze with mineral acids after about 16 hours'

boiling. Some proteins, the albuminoids and racemized proteins, resist

tryptic digestion but yield to acid hydrolysis. We know also that some

NH-CO linkages of proteins are broken down with great difficulty by

trypsin as it is difficult to obtain a tryptic digest of protein which

does not give the biuret reaction, and the work of Fischer and

Abderhalden has shown that certain artificial polypeptides are not

digested by pure activated pancreatic juice.

We have, then, three possibilities: Luciferin is (1) either a natural

proteose not attacked by trypsin, or (2) if attacked by trypsin its

decomposition products (presumably amino-acids) still contain the group

oxidizable with light production, or (3) it is not protein at all. I

have been unable to oxidize with light production various mixtures of

amino-acids (from tryptic digestion of beef and casein, or the acid

hydrolysis products of luciferin itself) by means of luciferase, and

consequently am led to believe that _Cypridina_ luciferin is either a

new natural proteose, soluble in absolute alcohol and not digested by

trypsin or that it belongs to some other group than the proteins. The

absence of a biuret reaction would point in that direction and the

question must await further study.

_Cypridina_ luciferin is found in the luminous gland of the animal and

possibly in parts non-luminous as well as in the luminous organ. This is

true of the luciferin from fireflies which is found throughout the body

of _Luciola_, _Photuris_ and _Photinus_.

CYPRIDINA LUCIFERASE.--Luciferase, on the other hand, has _all_ the

properties of a complex protein. It will not dialyze through collodion

or parchment membranes, is soluble only in aqueous solvents, and hence

precipitated by alcohol and acetone, digested by proteolytic enzymes,

readily changed by contact with dilute acid and alkali and irreversibly

coagulated on boiling. It is completely salted out of solution by

saturation with (NH_{4})_{2}SO_{4} and nearly completely precipitated by

the alkaloidal reagents. Its other properties are given in Table 8.

Taken together, they point to the group of albumins as the class of

proteins with which luciferase most closely agrees.

If luciferase is not a protein it is so closely bound up with protein

that it cannot be separated. This is characteristic of many enzymes and

luciferase is also an enzyme. We can determine this by finding out

whether luciferase will accelerate the oxidation of a large amount of

luciferin, for such is the test of a catalytic substance. If we take 1

c.c. of a dilute solution of luciferase (1 _Cypridina_ to 50 c.c. water)

and add to it successive 1 c.c. portions of concentrated luciferin (1

_Cypridina_ to 2 c.c. solution) as soon as the light from the preceding

addition has disappeared, after four 1 c.c. additions, no more light is

produced. The luciferase is therefore used up and cannot oxidize more

than a certain quantity of luciferin. In this experiment, however, we

added a concentration of luciferin from one _Cypridina_ 100 times that

of the luciferase from one _Cypridina_, i.e., four additions each 25

times as concentrated. We have, of course, no way of telling what the

absolute amount (in milligrams) of luciferin or luciferase is in a

single _Cypridina_, but we do know that the luciferase from one

_Cypridina_ cannot oxidize luciferin from more than 100 Cypridinas. If

the ratio of luciferin to luciferase in a single animal is 100:1, it

would mean that luciferase could oxidize 10,000 times its weight of

luciferin. A large excess of luciferin but not an indefinite quantity

can be oxidized by luciferase, and I believe this is sufficient

justification for considering luciferase an enzyme, although it is not

an ideal example of an organic catalyzer. Quite a number of enzymes are

known to be diminished during the course of the reaction they accelerate

or to be poisoned by their reaction products. Enzyme reactions inhibited

by the formation of reaction products again proceed if these are removed

or diluted. However, light does not again appear in a mixture of weak

luciferase with excess of luciferin upon dilution with water, so that

the luciferase cannot have been merely inhibited by some reaction

product but must have been actually used up during the reaction. It

should be noted in passing that the peroxidases, ordinarily spoken of as

oxidizing enzymes, are used up in the reaction and can only oxidize

limited amounts of oxidizable substances, a quantity almost in

proportion to the concentration of peroxidase present.

Whether luciferase is an oxidizing enzyme made up of an albumin

associated with some heavy metal as iron, copper or manganese is

uncertain. From analyses of whole _Cypridina_, kindly made for me by

Prof. A. H. Phillips of Princeton University, all three of these metals,

which we know to be associated with biological oxidations, are present,

and it is quite possible that one of them is concerned with the

oxidation of luciferin.

Although I have tested a great many oxidizers, organic and inorganic,

and a large number of oxidizing enzymes from blood and tissue extracts

of animals rich in iron, copper and manganese, I have found no material

which is capable of taking the place of _Cypridina_ luciferase.

Peroxidases or oxidases of plants, haemoglobin, haemocyanin, extracts of

mussels, manganese containing blood of various marine crustacea and

mollusks will give no light on mixing with luciferin. Such active

oxidizers as KMnO_{4}, H_{2}O_{2}, BaO_{2}, and many others, will not

oxidize _Cypridina_ luciferin with light production, although they can

oxidize _Pholas_ luciferin with light production.

The action of _Cypridina_ luciferase is very highly specific. It is

found only in the luminous organ of _Cypridina hilgendorfii_, not in

non-luminous parts and not in a non-luminous species of _Cypridina_

closely related to _hilgendorfii_.

Luciferins and luciferases from closely allied luminous forms will

mutually interact to produce light, but no light appears if these

substances come from distantly related forms. Thus firefly (_Photuris_)

luciferin will give light with _Pyrophorus_ luciferase and _vice versa_,

but _Cypridina_ luciferin will give no light with firefly (_Luciola_)

luciferase or _vice versa_, nor with _Pholas_ luciferase or _vice

versa_. The faint luminescences sometimes observed on mixing firefly or

_Cypridina_ luciferase with boiled extracts of non-luminous forms, or of

distantly related luminous forms, are probably caused by photophelein in

the boiled extract.

Like the plant peroxidases, _Cypridina_ luciferase is not readily

affected by the action of chloroform, toluol, etc. Unlike the plant

peroxidases, it will not oxidize (_i.e._, produce coloration) in either

presence or absence of H_{2}O_{2}, any of the hydroxyphenol or

aminophenol compounds, such as pyrogallol, a-naphthol,

para-diamino-benzine, gum guaiac, etc., commonly used as peroxidase

reagents. Neither will luciferase produce light with any substances,

such as oils, lophin, pyrogallol, gallic acid, esculin, etc., which we

know to be capable of oxidation with light production by other means.

The luciferases are very highly specific and act only upon the

luciferins of the same or closely related species. They must be placed

by themselves in a new class of oxidizing enzymes.

According to Dubois, _Pholas_ luciferase is rather readily destroyed by

chloroform and my own observations indicate that this is true also of

firefly luciferase, so that a certain amount of variation exists in the

group of luciferases.

None of the luminescent animals which I have studied are at all affected

by cyanides. The luminescence continues in extracts of _Cypridina_,

firefly, and _Cavernularia_, or in _Noctiluca_ and luminous bacteria

after addition of small or high (_m_/40) concentrations of KCN. In this

respect the luciferases are very different from many types of oxidizing

enzymes which are inhibited by exceedingly weak concentrations of

cyanide. It should be borne in mind, however, that while KCN inhibits

catalase and the catalytic decomposition of H_{2}O_{2} by Pt or Ag, it

does not affect the catalytic decomposition of H_{2}O_{2} by thallium.

OXYLUCIFERIN.--When luciferin is oxidized it must be converted into some

substance or substances and I believe this change involves no

fundamental destruction of the luciferin molecule as it is a reversible

process. I shall speak of the principal (if not the only) product formed

as _oxyluciferin_.

If we assume that the oxidation of luciferin changes the molecule but

slightly, we at once think of comparing the change luciferin <=>

oxyluciferin with the change reduced haemoglobin <=> oxyhaemoglobin. The

condition is, however, not so simple as this, for oxyhaemoglobin will

again give up its oxygen providing the partial pressure of oxygen is

made sufficiently low, whereas oxyluciferin will not do this, at least

in the dark. We can not reduce oxyluciferin solution by exhausting the

oxygen with an air-pump.

There is another oxidation-reduction system which can also be easily

reversed, but not by merely removing the oxygen from the solution--that

is, the reduction of a dye such as methylene blue to its leuco-base. I

believe the change which occurs when luciferin is oxidized is similar to

that which occurs when the leuco-base of methylene blue or sodium

indigo-sulphonate is oxidized to the blue dye. Oxidation of leuco-dye

bases occurs spontaneously in presence of oxygen and appears to consist

in the removal of hydrogen from the leuco-base with formation of water.

Reduction of these dyes may be effected in the same ways that

oxyluciferin can be reduced. In the case of methylene blue, reduction

consists in the addition of two hydrogen atoms. Whether a similar change

occurs when oxyluciferin is reduced or whether oxygen is actually added

as in formation of haemoglobin cannot be definitely stated at present. We

may write equations representing these possibilities as follows:

C_{16}H_{20}N_{3}SCl(leuco-methylene blue) + O <=> C_{16}H_{18}N_{3}SCl

(methylene blue) + H_{2}O

Haemoglobin + O <=> oxyhaemoglobin.

Let us now turn to the methods which may be used in reduction of

oxyluciferin. We may then endeavor to write an equation which will

represent the fundamental changes in the luminescence reaction.

My attempts to reduce the oxidation product of luciferin started from

the observation that if one places a clear solution of luciferase in a

tall test tube, although it may give off no light at first when shaken,

after standing a day or so a very bright light would appear on shaking.

This was especially true when the luciferase had become turbid and

ill-smelling from the growth of bacteria. Thinking that the bacteria

produced a substance which could be oxidized by the luciferase, I tried

growing bacteria and also yeast on appropriate culture media, and after

some days of growth mixing the culture media containing the products of

bacterial or yeast growth with luciferase, expecting to obtain light;

but no light appeared. However, if a little crude luciferase solution

was added to the bacterial or yeast cultures and then allowed to stand

for some hours, light appeared whenever they were shaken. Indeed such

cultures behaved much as a suspension of luminous bacteria which has

used up all the oxygen in the culture fluid and will only luminesce

when, by shaking, more oxygen dissolves in the culture medium.

Realizing that in bacterial cultures in test tubes, anaerobic

conditions soon appear, and also the strong reducing action of bacteria

upon many substances (for instance, nitrates or methylene blue) under

anaerobic conditions, it struck me that the bacteria might be reducing

the oxidation product of luciferin to luciferin again. We must remember

that since crude luciferase solution is a cold-water extract of a

luminous animal allowed to stand until all the luciferin has been

oxidized, it must contain oxyluciferin as well as luciferase and will

give light if the oxyluciferin is again reduced and oxygen admitted.

This appears to be the correct explanation of the above experiments.

Oxyluciferin may also be readily reduced by the use of the blood of the

horse-shoe crab (_Limulus_) allowed to stand until bacteria develop.

This experiment is of special interest because the blood contains

haemocyanin, which is colorless in the reduced condition and blue in the

oxy-condition. The color change thus serves as an indicator of the

oxygen concentration in the blood. A sample of foul-smelling _Limulus_

blood full of bacteria will become colorless on standing in a test tube

for 10 to 15 minutes, but the blue color quickly returns if shaken with

air. Such a blood has the power of reducing oxyluciferin through the

activity of the bacteria which it contains. Fresh blood has very little

if any reducing action.

Not only bacteria but also tissue extracts have a strong reducing action

in absence of oxygen. Thus, muscle tissue stained in methylene blue will

very quickly decolorize (reduce) the methylene blue if oxygen (air) is

kept away, but the blue color immediately returns if air is admitted.

Oxyluciferin (_i.e._, a solution of luciferin which has been completely

oxidized by boiling or standing in air until it no longer gives light

with luciferase) if mixed with a suspension of ground frog's muscle and

kept in a well-filled and stoppered test tube for some hours, is reduced

to luciferin and gives a bright light if now poured into luciferase

solution. Frog muscle suspension alone, or oxyluciferin alone, give no

light with luciferase, nor will a mixture of frog muscle suspension and

oxyluciferin, if shaken with air for several hours. Only if this last

mixture be kept under anaerobic conditions is the oxyluciferin reduced.

The reducing action of tissues is said to be due to a reducing enzyme

(_reducase_ or _reductase_), itself composed of a perhydridase and some

easily oxidized body such as an aldehyde. In the presence of the

perhydridase the oxygen of water oxidizes the aldehyde and the hydrogen

set free reduces any easily reducible substance which may be present.

There is a perhydridase in fresh milk, spoken of as _Schardinger's

enzyme_, which is destroyed by boiling. If some aldehyde is added, fresh

milk will reduce methylene blue to its leuco-base or nitrates to

nitrites, upon standing a short time. If shaken with air the blue color

returns. There is no reduction unless an aldehyde is added or unless

some boiled extract of a tissue such as liver is added. The boiled-liver

extract has no reducing action of its own, but supplies a substance

similar to the aldehyde which has been spoken of as _co-enzyme_. The

aldehyde is oxidized to its corresponding acid. Milk will reduce

methylene blue without aldehyde if bacteria are present in large

numbers. There is no reduction if the milk, methylene blue, and aldehyde

are agitated with air. The temperature optimum is rather high, 60 deg. to

70 deg. C.

I find that milk is a favorable and convenient medium for the reduction

of oxyluciferin and that it acts without the addition of an aldehyde or

the presence of bacteria. There is probably a substance acting as the

aldehyde in the luciferase-oxyluciferin solution. No light appears if

milk is added to a luciferase-oxyluciferin solution, but if the mixture

is allowed to stand in absence of oxygen light will appear when air is

admitted. The air can be conveniently kept out by filling small test

tubes completely with the solution and closing them with rubber

stoppers.

As almost all animal tissues contain reductases it is not surprising to

find that a freshly prepared and filtered extract of _Cypridina_

containing oxyluciferin and luciferase, which gives no light on shaking,

will, on standing in a stoppered tube for 24 hours at room temperature in

the dark give light when air is admitted. While this may be due to the

development of bacteria with a reducing action, it does not seem likely,

as under the same conditions methylene blue is not reduced in 24 hours,

and there is no turbidity or smell of decomposition in the tube. In 48

hours bacteria appear and methylene blue is also reduced. If we add

chloroform, toluol or thymol to the tubes of _Cypridina_ extract to

prevent the growth of bacteria, and allow them to stand 48 hours, upon

admitting air the tube with chloroform gives no light but the tubes with

toluol and thymol do give light, although it is not so bright as if they

were absent. I believe that these substances have a destructive action on

the reductases, most complete in the case of chloroform. Dubois (1919_c_)

also has recorded the occurrence of a reducing enzyme in _Pholas_, a

"hydrogenase," which is able to form hydrogen from cane sugar, and

luciferin from a boiled extract of _Pholas_. He now regards it as

identical with his co-luciferase.

I have not been able to demonstrate that a _Cypridina_ extract will

reduce methylene blue, or nitrates to nitrites, either with or without

the addition of acetaldehyde. This may be due to the fact that

oxyluciferin, which is also present, may be reduced more readily than

either nitrates or methylene blue, and so is reduced first.

We can also reduce oxyluciferin by means which do not involve the use of

animal extracts. Perhaps the best of these is reduction by palladium

black and sodium hypophosphite. The latter is oxidized in presence of

palladium and nascent hydrogen is set free. The nascent hydrogen reduces

any easily reducible substance which may be present, such as methylene

blue or oxyluciferin. Oxyluciferin is not reduced by palladium alone or

hypophosphite alone, but methylene blue is reduced by palladium black

alone.

If hydrogen sulphide is passed through a solution of methylene blue the

dye is very quickly reduced and becomes colorless. If the H_{2}S is

driven off by boiling the colorless methylene-blue solution, the blue

color again returns on cooling. Oxyluciferin can also be reduced by

H_{2}S.

If one adds some Mg powder to oxyluciferin and then dilute acetic acid

in successive additions as the acetic acid is used up in formation of Mg

acetate, the oxyluciferin will be reduced relatively quickly. Nascent

hydrogen is produced in the reaction and is no doubt the active reducing

agent.

Dilute acid favors the reduction of oxyluciferin. If one saturates an

oxyluciferin solution with CO_{2} or adds a little dilute acetic acid,

HCl, HNO_{3} or H_{2}SO_{4}, to it, a certain amount of reduction will

occur. No reduction occurs if the solution is saturated with pure

hydrogen, even if allowed to stand 24 hours. The action of the acid

begins when the solution of oxyluciferin, ordinarily slightly alkaline

(PH = 9), is made neutral (PH = 7.1) as indicated in Table 9. The action

of the acid must be on the oxyluciferin, as no luciferin or other

enzymes destroyed on boiling are present.

TABLE 9

_Effect of Acid on Reduction of Oxyluciferin_

=============================================================================

Solution | P_{H}|Luminescence| Remarks

| | with |

| | luciferase |

-------------------------------+------+------------+-------------------------

20 c.c. Oxyluciferin alone | 9.01 | Negative |

20 c.c. Oxyluciferin + .05 c.c.| 8.8 | Negative |

5 per cent. acetic acid | | |

20 c.c. Oxyluciferin + .15 c.c.| 7.1 | Fair |

5 per cent. acetic acid | | |

20 c.c. Oxyluciferin + .30 c.c.| 5.9 | Good |Acid forms precipitate in

5 per cent. acetic acid | | | this oxyluciferin sol.

20 c.c. Oxyluciferin + .50 c.c.| | Good |Acid forms precipitate in

5 per cent. acetic acid | | | this oxyluciferin sol.

20 c.c. Oxyluciferin + .75 c.c.| | Good [10] |Acid forms precipitate in

5 per cent. acetic acid | | | this oxyluciferin sol.

-------------------------------+------+------------+-------------------------

[10] Light disappears quickly because of the effect of the acidity on

the luciferase.

It is possible that the action of bacteria (which produces CO_{2}),

muscle tissue (which contains lactic acid), milk (in which lactic acid

may be formed by bacteria), or Mg + acid, in forming luciferin, is not

the result of their reducing power but of their acidity. Fortunately we

can test this matter by the use of reducing fluids which are not acid.

If they also form luciferin from oxyluciferin, a reduction must occur.

Nascent H can be generated by the action of NaOH on Al, or when finely

divided Mg or Zn or Al is placed in water. With Mg the water becomes

only slightly alkaline from formation of almost insoluble Mg(OH)_{2}. If

we add some Al powder and dilute NaOH to an oxyluciferin solution, H is

given off and luciferin is formed. As oxyluciferin cannot be formed by

the addition of alkali alone we must have in this experiment a reduction

of oxyluciferin in alkaline medium by the nascent H produced. Luciferin

can also be formed by merely adding Al or Zn or Mg dust to an

oxyluciferin solution. Methylene blue can also be readily reduced to its

leuco-base by Zn dust or Al + NaOH.

Indeed, if one adds some Al or Zn or Mg powder to a solution of

luciferase, light will appear whenever the solution is shaken.

Luciferase solution must always contain the oxidation product of

luciferin, oxyluciferin. In presence of nascent H this is reduced to

luciferin, and since the reaction of the medium is alkaline and

luciferase is present this is oxidized with light production, when, by

shaking, air is dissolved. The light can never become very bright except

at the surface because of the deficiency of oxygen in the solution. It

would seem, then, that the action of bacteria, yeast, muscle cells,

etc., on oxyluciferin must be due not entirely to their acid reaction

but to their reducing power as well.

The above experiment is a very striking and instructive one. Given a

test tube of luciferase solution containing, as it does, oxyluciferin,

add some Zn dust or Mg powder, and the evolution of hydrogen begins.

Conditions are now favorable for the reduction of oxyluciferin and this

occurs. Shake the contents of the tube to dissolve oxygen and light

appears. Allow the tube to stand and the light soon disappears. Shake

again and the light reappears. The luminescence reduction and oxidation

process can be demonstrated many times.

A similar experiment can be performed with luciferase and oxyluciferin

solution by addition of NH_{4}SH. This will serve also as another

example of the reduction of oxyluciferin in an alkaline medium. Whenever

we shake a tube of luciferase, oxyluciferin and NH_{4}SH, light will

appear. When the tube is at rest it becomes dark. Even the merest touch

is sufficient to agitate the tube contents, cause solution of oxygen and

appearance of light. It is just as if we stimulate the tube to produce

light and I believe the phenomenon has a deeper significance and a more

fundamental similarity to the phenomena of stimulation than may at first

appear. What more simple means of controlling a process can we think of

than by admission or withdrawal of oxygen? The firefly turns on its

light by stimulation through nerves of the luminous organ. _Noctiluca_

flashes on stimulation of any kind, even the slightest agitation causing

a brilliant emission of light. If the stimulation process means merely

the admission of oxygen to the photogenic cells we have a mechanism in

the cell itself for automatically producing the light. The admission of

oxygen results in aerobic conditions and luciferin in presence of

luciferase can then oxidize to oxyluciferin with luminescence. When the

oxygen is used up, the light ceases, anaerobic conditions prevail, and

the oxyluciferin is reduced to luciferin again. Thus, luciferin is

reformed during the rest period of _Noctiluca_ or between the flashes of

the firefly. What more efficient type of light than this is to be

desired?

Again, methylene blue offers an interesting parallel to oxyluciferin. A

little NH_{4}SH added to methylene blue solution will reduce

(decolorize) it to the leuco-base. If the tube is now shaken the blue

color returns. On standing reduction again occurs. The process can be

repeated a number of times, the reaction going in one or the other

direction, depending on the oxygen content of the mixture.

As methylene blue contains no oxygen, its reduction consists in the

addition of two atoms of hydrogen. When leuco-methylene blue oxidizes,

water is formed by the union of these two atoms of hydrogen with oxygen,

thus:

C_{16}H_{20}N_{3}SCl + O <=> C_{16}H_{18}N_{3}SCl + H_{2}O

(leuco-methylene blue) (methylene blue)

Briefly--MH_{2} + O <=> M + H_{2}O

To reduce methylene blue we can add the two hydrogen atoms directly from

nascent hydrogen formed in the solution or we can split up water by a

catalyzer in the presence of some substance, which will take up the

oxygen of water, thus:

NaH_{2}PO_{2} + H_{2}O + Pd = NaH_{2}PO_{3} + H_{2} + Pd

(Sodium hypophosphite) (Sodium phosphite)

This reaction occurs in presence of finely divided palladium. Methylene

blue can be reduced by the H_{2} and the hypophosphite oxidized.

Since oxyluciferin can be reduced by palladium and sodium hypophosphite

(Harvey, 1918), it is probable that we can write the equation for

reduction of oxyluciferin and oxidation of luciferin in a similar manner

to that of methylene blue:

Luciferin + O <=> Oxyluciferin + H_{2}O

Briefly--LH_{2} + O <=> L + H_{2}O.

Just as in the case of methylene blue the reaction proceeds in the right

hand direction spontaneously if the pressure of O is sufficiently high.

If luciferase is also present we have luminescence.

LH_{2} + O + luciferase <=> L + H_{2}O + luciferase (luminescence)

The reaction proceeds in the left hand direction under low oxygen

pressure, in the presence of nascent hydrogen or with some catalyzer

which is able to split water, transferring the H_{2} to oxyluciferin and

the O to an acceptor (A). NaH_{2}PO_{2} plays the part of the acceptor.

L + H_{2}O + A + Pd = LH_{2} + AO + Pd.

This appears to be the way in which the reducing enzymes or

perhydridases (comparable to the Pd) of living tissues act (Bach,

1911-13) and the action of yeast cells, bacteria, muscle suspensions,

etc., in reducing oxyluciferin must occur in the same manner.

If we assume that the LH_{2} (luciferin) compound is dissociated to even

the slightest extent into L and hydrogen, the hydrogen ion will shift

the equilibrium toward the formation of that substance which involves

the taking up of hydrogen. Consequently we may obtain a partial

formation of luciferin by adding an acid to oxyluciferin. Reduction of

the H-ion concentration tends to shift the equilibrium in the opposite

direction. Consequently, addition of alkali favors the oxidation of

luciferin, and it is quite generally true that biological oxidations are

favored by an alkaline reaction. In addition oxygen in alkaline medium

has a higher oxidation potential than in neutral or acid media. I

believe this is the explanation of the action of acid in formation of

luciferin from oxyluciferin.

Addition of acid is not the only means of favoring the formation of

luciferin from oxyluciferin. Any reaction which proceeds in one

direction with evolution of light should, theoretically, proceed in the

opposite direction under the influence of light. So far as I know the

case of a reaction, photogenic in one direction and photochemical in the

other direction, has never been described, unless we are to accept the

cases of phosphorescence, for instance, the absorption of light by CaS

and its emission in the dark. However, the reaction which occurs during

phosphorescence cannot be stated.

It is a fact that light will cause the reduction of oxyluciferin. A tube

of oxyluciferin exposed to sunlight for six hours, or the mercury arc

for two hours, will be partially converted into luciferin. It will

luminesce when luciferase is added, while a control tube kept in

darkness shows no trace of luciferin. The action is more marked with the

ultra-violet as a solution of oxyluciferin in a quartz tube showed more

reduction than one in a glass tube when exposed for the same length of

time to the quartz mercury arc. The reduction is not dependent on the

formation of acid under the influence of light since two tubes of

oxyluciferin, one kept in darkness and the other exposed to sunlight for

six hours, had the same reaction, PH = 9.3. Of course some reducing

substance might be formed under the influence of light but this is not

very probable.

We may therefore write the reaction for luminescence in the following

way:

darkness

alkali

luciferase

luciferin (LH_{2}) + O <=> oxyluciferin (L) + H_{2}O (luminescence)

perhydridase

acid

light

Acid and light favor reduction while alkali and darkness favor oxidation

in the luciferin <=> oxyluciferin reaction. Whether the luciferin be really

oxidized by removal of H_{2} or whether by addition of oxygen is, of

course, uncertain, but the analogy with methylene blue is striking and

may serve as a working hypothesis until the composition of luciferin and

its oxidation product are known.

While I have not studied the properties of oxyluciferin as fully as

those of luciferin, so far as I can judge, both substances give the same

general reactions and possess identical properties. Both crude luciferin

and crude oxyluciferin solution are yellow in color, but I do not

believe that either pure luciferin or oxyluciferin are yellow in color,

because an ether or benzine extract of _Cypridina_ is also yellow,

although luciferase, luciferin, and oxyluciferin are insoluble in ether

and benzine. The yellow pigment which can be observed to make up part of

the luminous gland of _Cypridina_ is not luciferin or luciferase. It may

be a pigment related to _urochrome_.

When tests are applied and precipitating reagents are added to crude

luciferin and crude oxyluciferin solution, they give identical results

in each case. A more complete account of the chemistry of luciferin has

been given in this chapter, and there is no need of duplicating these

statements regarding oxyluciferin. Like luciferin, the oxyluciferin will

pass porcelain filters, dialyze through parchment or collodion

membranes, and is undigested by salivary diastase, pepsin HCl, Merck's

pancreatin in neutral solution, and erepsin. The salivary diastase and

the pancreatin (containing amylopsin, trypsin, and lipase) were allowed

to digest for four days at 38 deg. C. without showing any evidence of

digestive action.

As luciferin is so easily oxidizable a substance, we should expect to

find that it will reduce just as glucose will reduce. However, a

concentrated solution of luciferin has no reducing action on Fehling's

(alkaline Cu), Barfoed's (acid Cu), Nylander's (alkaline Bi) or Knapp's

(alkaline Hg) reagent. Glucose will reduce methylene blue in alkaline

(not in neutral solution), but luciferin will not reduce methylene blue

in alkaline or neutral solution. It would seem, then, that luciferin

must contain no aldehyde group. If so, we should expect to obtain

reduction of some of the above reagents. Just what group is concerned in

the oxidation is unknown at the present time, and in the absence of more

experimental data, speculation regarding it can be of little value.

SUMMARY

In summing up we may say that the luminescence of at least three groups

of luminous animals, the beetles, _Pholas_, and _Cypridina_, has been

definitely shown to be due to the interaction of two substances,

luciferin and luciferase, in presence of water and oxygen. Luciferin and

luciferase have quite different properties and may be easily separated

from each other by various chemical procedures. As the luciferins and

luciferases from different luminous animals have somewhat different

properties, they may be designated by prefixing the generic name of the

animal from which they are obtained.

_Cypridina_ luciferin differs from _Pholas_ luciferin in that it can not

be oxidized with light production by KMnO_{4}, H_{2}O_{2}, with or

without haemoglobin, or similar oxidizing agents. _Cypridina_ luciferase

differs from _Pholas_ and firefly luciferase in that it is not readily

destroyed by the fat-solvent anaesthetics, such as chloroform, ether,

etc.

When _Cypridina_ luciferin is oxidized, no fundamental splitting of the

molecule occurs, because the product, oxyluciferin, can be readily

reduced to luciferin again. This reduction is brought about under

conditions similar to those necessary for the reduction of dyes, such as

methylene blue. Oxyluciferin can be reduced to luciferin, which will

again give light with luciferase, by the reductases of muscle tissue,

liver, etc., or by bacteria; by Schardinger's enzyme of milk; by H_{2}S;

by the nascent hydrogen from the action of acetic acid on magnesium or

of water or NaOH on aluminium, zinc or magnesium; and by palladium black

and sodium hypophosphite, all well-known reducing methods. Reduction of

oxyluciferin no doubt occurs even in presence of luciferase if oxygen is

absent, and reduction of oxyluciferin no doubt occurs in animals which

burn luciferin within the cell, thus tending for conservation of

material. Dilute alkali favors oxidation and dilute acid favors the

reduction. Light favors the reduction of oxyluciferin.

Apparently luciferin and oxyluciferin have identical chemical

properties. Neither is digested by the enzymes: malt diastase, ptyalin,

yeast invertase, pepsin, trypsin, steapsin, amylopsin, rennin, erepsin,

urease or enzymes occurring in the water extracts of dried spleen,

kidney, or liver. Luciferase is destroyed only by pepsin (probably),

trypsin, erepsin, and something in spleen and liver extract.

Luciferase is unquestionably a protein and all its properties agree with

those of the albumins. Although used up in oxidizing large quantities of

luciferin, it behaves in many ways like an enzyme and may be so

regarded.

Luciferin, on the other hand, is not digested by proteolytic enzymes, is

dialyzable, almost but not completely precipitated by saturation with

(NH_{4})_{2}SO_{4}, and is soluble in absolute alcohol, acetone, and

some other organic solvents, but not in the strictly fat-solvents like

ether, chloroform, and benzol. There are, however, certain CO-NH

linkages which are not attacked by proteolytic enzymes and some peptones

soluble in absolute alcohol, so that these two characteristics do not

bar it from the group of proteins. Luciferin, in fact, has many

properties in common with the proteoses and peptones but its chemical

nature cannot be definitely stated at present.

CHAPTER VII

DYNAMICS OF LUMINESCENCE

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 [alpha] 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 phosphorescent 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

assuming 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 x 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 x 10^{-10}

ergs if completely oxidized. This quantity 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 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 deg. C. To put it in the

usual way, its temperature coefficient (Q_{10}) for a 10 deg. 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 deg. C. practically no light is produced by many

oxidizers which are themselves unaffected at 100 deg.. 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 deg. | 20 deg. | 50 deg. | 75 deg. | 98-100 deg.

----------------------+----------+---------+---------+--------+----------

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

M/100 KMnO_{4} | Fair | Good | Bright | Bright | Faint

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

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

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

MnO_{2} | Negative | Fair | Fair | Fair | Negative.

----------------------+----------+---------+---------+--------+----------

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 deg. and increases 13.19 mm. Hg for each

degree rise in temperature. This means that for a definite temperature,

say, 20 deg., 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 analogous 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, passed

through an ozonizer (the silent electric discharge tube), is bubbled

through m/100 pyrogallol, no luminescence occurs at 0 deg., a fair

luminescence at 20 deg., a good luminescence at 50 deg., and a bright

luminescence at the boiling point. If the pyrogallol is of _m_

concentration, no luminescence occurs at 0 deg. or 20 deg., a fair luminescence

at 50 deg., and a bright luminescence at the boiling point. For a definite

temperature, say 20 deg., 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 assume 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. Assuming 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 mass 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 deg. | 10 deg. | 20 deg. | 30 deg. | 50 deg. | 75 deg. | 98-100 deg.

--------+---------+---------+-------+--------+--------+--------+---------

| | | | | | |

--------+---------+---------+-------+--------+--------+--------+---------

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 deg. | 10 deg. | 20 deg. | 30 deg. | 50 deg. | 75 deg. |98-100 deg.

--------------------+---------+-----+-----+-----+------+--------+-------

--------------------+---------+-----+-----+-----+------+--------+-------

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 Potassium ferrocyanide | - |Bright| + | | +

(K_{4}Fe(CN)_{6} M/10-M/20) | | | | |

2 Potassium ferricyanide | - |Very | - | - |Very

(K_{3}Fe(CN)_{6} M/10-M/1,250) | | faint| | | slow

| | to - | | |

3 Potassium chromate | - |Good | + | | +

(K_{2}CrO_{4} M/20-M/100) | | | | |

4 Potassium bichromate | - |Good | + | | +

(K_{2}Cr_{2}O_{7} M/50-M/100) | | | | |

5 Potassium permanganate | - |Bright| + | - | +

(KMnO_{4} M/50-M/200) | | | | |

6 Potassium hydroxide | - | - | - | - |Very

7 Potassium chlorate | - | - | - | - | -

(KClO_{3} M/10) | | | | |

8 Potassium persulfate | - | - | - | - | -

(K_{2}S_{2}O_{8} M/10-M/128) | | | | |

(Cr_{2}(SO_{4})_{3}.K_{2}SO_{4} M/10) | | | slow| slow|

(Fe_{2}(SO_{4})_{3}.(NH_{4})_{2}SO_{4} | | | | | slow

M/10) | | | | |

(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 | - | - | + | | +

21 Sodium hypoiodite | - |Faint | + | | +

22 Calcium hypochlorite | - |Good | + | | ++

(Ca(OCl)_{2} saturated solution) | | | | |

23 Turnip juice | - |Bright| - | + | ++

24 Turnip juice heated to 70 deg. | - |Faint | - | + |Very

25 Turnip juice boiled | - | - | - | - | -

26 Albumin solution | - | - | - | - | -

27 Albumin solution + KMnO_{4} | - |Good | + | - | ++

28 Albumin 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) | - | - | - | - | -

Contains hemocyanin | - |Fair | | | ++

39 Squid blood (Sepia esculenta) boiled | - |Good | | | -

tetronerythrin, a lipochrome | - |Faint | | | ++

boiled | - |Fair | | | -

43 Annelid blood (Laonome japonica) boiled | - | - | | |

(Cavernularia haberi) | - | - | - | + | ++

(Cypridina hilgendorfii) | - | - | | | +

(Noctiluca miliaris) | - | - | - | - | -

luminous organs | - | - | | | ++

48 Ferrous ferrocyanide (Fe_{2}Fe(CN)_{6}) | - |Faint | + | | +

49 Zinc ferrocyanide (Zn_{2}Fe(CN)_{6}) | - | - | + | |Very

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 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

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 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 deg. (depending on various

conditions), where coagulation of luciferase occurs. Light will appear

at 0 deg. 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 deg. 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 deg. 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.

TABLE 14

_Temperature Limits of Luminescence for Luminous Organism_

-------------------------+---------------------+-------+-------+-------+

-------------------------+---------------------+-------+-------+-------+

Pseudomonas javanica | Eijkman, 1892 | -20 deg. | 25-33 deg.| 45 deg. |

| | | | |

Bacterium phosphorescens | Lehmann, 1889 | -12 deg. | ... | 39.5 deg. |

| | | | |

Bacterium phosphoreum | Molish, 1904, book | -5 deg. | 16-18 deg.| 28 deg. |

| | | | |

Light bacteria | Tarchanoff, 1902 | -7 deg. | 15-25 deg.| 37 deg. |

| | | | |

Light bacteria | Harvey, E. N., 1913 | -11.5 | 15-20 deg.| 38 deg. |

| | | | |

Mycelium X | Molish, 1904 | ... | 15-25 deg.| 36 deg. |

| | | | |

Lampyrids | Macaire, 1821 | -10 | 33 deg. | 46-50 deg.|

| | | | |

Pyrophorus noctilucus | Dubois, 1886 | ... | 20-25 deg.| 47 deg. |

| | | | |

Photuris pennsylvanica | Lund, 1911 | ... | ... | 50 deg. |

| | | | |

Luciola viticollis | Harvey, E. B., 1915 | <0 deg. | ... | 42 deg. |

| | | | |

Cypridina hilgendorfii | Harvey, E. N., 1915 | <0 deg. | ... | 52-54 deg.|

| | | | |

Cyclopina gracilis | Lund, 1911 | ... | ... | 50 deg. |

| | | | |

Phylirrhoe bucephalum | Panceri, 1872 | 44 deg. | ... | 61 deg. |

| | | | |

Pyrosoma | Panceri, 1872 | <0 deg. | ... | 60 deg. |

| | | | |

Mnemiopsis Leidyi | Peters, 1905 | 9 deg. | 21 deg. | 37 deg. |

| | | | |

Noctiluca miliaris | Quatrefages, 1850 | 1 deg. | ... | 40 deg. |

| | | | |

Noctiluca miliaris | Harvey, E. B., 1917 | <0 deg. | ... | 48 deg. |

| | | | |

Cavernularia haberi | Harvey, E. N., 1915 | <0 deg. | ... | 52 deg. |

| | | | |

Watasenia scintillans | Shoji, R, 1919 | ... | 16-31 deg.| 49 deg. |

-------------------------+---------------------+-------+-------+-------+

We are thus led to the conclusion that intensity of luminescence is

dependent on the velocity of oxidation of luciferin and that with

lowered reaction velocity the spectral composition of the light changes.

The maximum emission shifts toward the yellow. I believe, however, that

in _Cypridina_ also, the luminescence intensity depends not only on

reaction velocity but on the particular manner in which luciferin is

oxidized. _Cypridina_ luciferin will luminesce only in presence of

_Cypridina_ luciferase and no light can be obtained from _Cypridina_

luciferin and a host of different oxidizers (with or without H_{2}O_{2})

such as are able to oxidize pyrogallol. Luciferin will also oxidize in

the air spontaneously but no light is produced. It is easy to show that

this spontaneous oxidation may be much more rapid than an oxidation with

luciferase and yet light appears only in presence of the latter. If a

concentrated solution of luciferin is kept near the boiling point it

will be completely oxidized to oxyluciferin in four or five minutes. No

light appears if air or even if pure oxygen is bubbled through it. The

same solution kept at 20 deg. with a small amount of luciferase will

luminesce continuously and not be completely oxidized to oxyluciferin in

a half hour. We can, however, cause the luciferin to oxidize as rapidly

at 20 deg. by adding concentrated luciferase as does the luciferin near the

boiling point without luciferase. A bright light is produced in the

former case, none in the latter case. The oxyluciferin formed from

spontaneous oxidation of luciferin appears to be the same as that formed

with luciferase present. Both give luciferin again on reduction. Perhaps

the reaction takes place in two stages, similar to those supposed to

occur in other enzyme actions:

luciferin + luciferase = luciferinluciferase

luciferinluciferase + O (or minus H_{2}) = oxyluciferin

+ luciferase.

We may then assume as a tentative hypothesis that luminescence only

occurs during oxidation (addition of O or removal of H) of the

luciferinluciferase compound.

We have just seen that the effect of cooling a _Cypridina_ extract

containing luciferin and luciferase and luminescing with a bluish light,

is to reduce the intensity and change the shade toward the yellow.

Velocity of oxidation must be lowered and with the same concentration of

luciferase lowered velocity means more light of the longer wave-lengths.

A very instructive experiment on color of the light can be carried out

with animals having different colored lights and so closely related that

their luciferins and luciferases will interact with each other. Such a

case is presented by the American fireflies, _Photinus_ and _Photuris_.

_Photinus_ emits an orange light, while _Photuris_ emits a greenish

yellow light. The difference in color is especially noticeable when the

luminous organs of the two forms are ground up in separate mortars. As

shown by Coblentz, the difference in color is real, the spectrum of

_Photinus_ extending farther into the red than that of _Photuris_ (see

Fig. 8). We can easily prepare luciferin and luciferase from the two

fireflies and make the following mixtures:

_Photinus_ luciferin x _Photinus_ luciferase = reddish light.

_Photinus_ luciferin x _Photuris_ luciferase = yellowish light.

_Photuris_ luciferin x _Photuris_ luciferase = yellowish light.

_Photuris_ luciferin x _Photinus_ luciferase = reddish light.

Thus the color of the light in these "crosses" is that characteristic of

the animal supplying the luciferase. To bring this fact in line with

what we have already said regarding reaction velocity and luminescence,

we must believe that the _Photinus_ luciferase oxidizes at a slower rate

than the _Photuris_ luciferase. In this connection it is of interest to

recall that the _Photuris_ light as emitted by the insect becomes

reddish at high temperatures, or if the insect is plunged into alcohol,

both conditions which bring about partial coagulation of the luciferase

and reduce its active mass.

When the dried powdered luminous material of an ostracod is sprinkled