The Nature of Animal Light Part 10

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 a.s.sume 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 phosph.o.r.escence, for instance, the absorption of light by CaS and its emission in the dark. However, the reaction which occurs during phosph.o.r.escence 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 gla.s.s 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 a.n.a.logy 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 pa.s.s 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 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 alb.u.mins. Although used up in oxidizing large quant.i.ties 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