The Nature of Animal Light Part 9

_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 cla.s.ses 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 cla.s.s 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 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 quant.i.ty 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 C., it does hydrolyze with mineral acids after about 16 hours'

boiling. Some proteins, the alb.u.minoids 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 alb.u.mins as the cla.s.s 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 quant.i.ty 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 quant.i.ty 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 pa.s.sing 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 quant.i.ty almost in proportion to the concentration of peroxidase present.

Whether luciferase is an oxidizing enzyme made up of an alb.u.min a.s.sociated with some heavy metal as iron, copper or manganese is uncertain. From a.n.a.lyses 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 a.s.sociated 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 cla.s.s 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 princ.i.p.al (if not the only) product formed as _oxyluciferin_.

If we a.s.sume 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 + O ? C_{16}H_{18}N_{3}SCl + H_{2}O (leuco-methylene blue) (methylene blue)

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 to 70 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 pa.s.sed 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.