Luciferase

“ 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. ”

“ 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. ”

“ 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° C., is non-dialyzable, insoluble in fat solvents, but forms a colloidal solution in water. It is not affected by 1 per cent. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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. ”

“ 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 (?) ”

“ 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. ”

“ 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. ”

“ The use of a living animal for such measurements introduces a possible source of error in that any contraction of the muscles of the animal will produce heat which may add to an increase or mask a decrease of temperature during luminescence. Utilization of extracts of luminous animals containing the luciferin and luciferase mentioned above avoids the complications due to muscular contraction. ”

“ 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. ”

“ 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 H2O2, 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 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. ”

“ 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. ”

“ He considers that the light is produced by the reactions of two special substances, luciferase and luciferine. Luciferase is of the nature of an enzyme, and exists only in the luminous organs, in the form, it is supposed, of extremely minute granules. ”

“ Whenever we shake a tube of luciferase, oxyluciferin and NH4SH, 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 aërobic conditions and luciferin in presence of luciferase can then oxidize to oxyluciferin with luminescence. ”

“ The fact that the putative Hermodice carunculata luciferase shows highest homology to the luciferase of a phylogenetically distant cnidarian (Renilla reniformis) can probably be attributable to the lack of publicly available luciferase sequences from more closely related organisms. The transcriptomic dataset presented herein can greatly help identify and characterize this putative photoprotein and facilitate future studies investigating the genetic and biochemical basis of light production in annelids. ”

“ 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 [Pg 123] 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. ”

“ 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 anaërobic 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. ”

“ 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. ”

“ 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. ”

“ We have seen that Bioluminescence is an oxylumines [Pg 111] cence, 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. ”

“ 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 [Pg 127] catalytic decomposition of H2O2 by Pt or Ag, it does not affect the catalytic decomposition of H2O2 by thallium. ”

“ I asked, much interested.
"I mean the book had some effect."
"Oh, I beg your pardon—of course, of course."
"But we want something more."
"Yes, we do," said I.
"What precisely it is," said Mrs. Knight, knitting her arched brows, "I am not quite sure. What is the next thing to be done, Mr. Vansittart?"
"To put in action," said I, firmly and without a moment's hesitation, "the gospel which you have so nobly preached in Bluesifera.'
"Forgive me—Lucifera, Mr. Vansittart."
"Yes, yes—Lucifera, I said Lucifera didn't I? At any rate, I meant—I say, what are you getting up for, Mrs. Knight?" ”

“ Mrs. Knight went on, with a swing of her fan through the air. "You men don't know what the deadening weight of it is. It is we women who have to bear the burden of it. We are trammelled and tied at every step."
"It's a burning shame," said I. "Why do you stand it?"
"Well, I have lifted up my voice," said Mrs. Knight. "I have claimed the right for myself and my sisters to do what our feelings—our feelings, which are nature's guide—tell us; as I say in Lucifera, through my heroine's lips." ”