The recording of ATP in the erythrocytes using luciferase injected into the cells

The luciferase preparation obtained from fireflies Luciola mingrelica has entrapped into the human erythrocytes by means of reversible osmotic lysis. The addition of luciferin to such erythrocytes leads to the appearance of luminescence, conditioned by the entrance of luciferin into the cells. Luciferin is uniformly distributed between cells and external medium. Luciferin transport through the erythrocyte membrane is a result of simple diffusion. Values of rate constant of luciferin transport through the membrane lie between 0.009-0.021 l/s 1 cells for erythrocytes of different donors. The maximum luminescence intensity increases monotonously with rise of temperature and luciferin concentration. The dependence of the maximum luminescence intensity on luciferin concentration is described by Michaelis kinetics. Obtained in different experiments, values of luciferase Michaelis constant for luciferin inside erythrocytes lie between 4.1-21.5 microM. Luminescence intensity of the luciferase containing erythrocytes depends on the intracellular ATP concentration. Under the same luciferin concentration the correlation of luminescence intensities of control erythrocytes with normal ATP level and erythrocytes depleted without glucose is near to correlation of their ATP concentrations. After the addition of glucose to the depleted erythrocytes their ATP concentration rises and luminescence intensity approaches to the level of control erythrocytes. Luciferase entrapment permit one to control rapid ATP concentration changes in the erythrocytes.     

ON THE QUANTA OF LIGHT PRODUCED AND THE MOLECULES OF OXYGEN UTILIZED DURING CYPRIDINA LUMINESCENCE

A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10^–5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10^–5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O₂ = oxyluciferin + H₂O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration.     

THE OXIDATION-REDUCTION POTENTIAL OF THE LUCIFERIN-OXYLUCIFERIN SYSTEM

The oxidation-reduction potential of the Cypridina luciferin-oxyluciferin system determined by a method of "bracketing" lies somewhere between that of anthraquinone 2-6-di Na sulfonate (E_o ^'' at pH of 7.7 = –.22) which reduces luciferin, and quinhydrone (E_o ^'' at pH of 7.7 = +.24), which oxidizes luciferin. Systems having an E_o ^'' value between –.22 and +.24 volt neither reduce oxyluciferin nor oxidize luciferin. If the luciferin-oxyluciferin system were truly reversible considerable reduction and oxidation should occur between –.22 and +.24. The system appears to be an irreversible one, with both "apparent oxidation" and "apparent reduction potentials" in Conant''s sense. Hydrosulfites, sulfides, CrCl₂, TiCl₃, and nascent hydrogen reduce oxyluciferin readily in absence of oxygen but without luminescence. Luminescence only appears in water solution if luciferin is oxidized by dissolved oxygen in presence of luciferase. Rapid oxidation of luciferin by oxygen without luciferase or oxidation by K₃Fe(CN)₆ in presence of luciferase but without oxygen never gives luminescence.     

Cytoplasmic factors that affect the intensity and stability of bioluminescence from firefly luciferase in living mammalian cells

In order to improve calibration of firefly luciferase signals obtained by injecting the enzyme into single, isolated heart and liver cells we have investigated why the luminescence from cells is greatly depressed compared with in vitro (in mammalian ionic milieu) and why the decay of the intracellular signal is remarkably slow. We have shown that inorganic pyrophosphate greatly depresses the signal in vitro and that micromolar concentrations of inoragnic pyrophosphate, comparable with that in cytoplasm, reverse this inhibition and stabilize the signal, eliminating its decay. Higher concentrations of pyrophosphate depress the signal by inhibiting ATP-binding to luciferase. Luciferse-injected cells exposed to extracellular luciferin concentrations above about 100 μmol/1 (corresponding to a cytoplasmic level of c. 5–10 μmol/1 because of a transplasmalemmal gradient) show a gradual, irreversible loss of signal. We attribute this phenomenon (which is not seen in vitro) to the gradual accumlation of a luminescently inactive, irreversible, luciferase-oxyluciferin complex. At low luciferin levels this complex is prevented from forming by cytoplasmic pyrophosphate. Above c. 100μmol/1 extracellular luciferin, the pyrophosphate level in the cytoplasm fails to fully prevent the complex forming. In vitro this phenomenon does not occur because the luciferase concentrations and hence oxyluciferin levels are orders of magnitude lower than in cells injected with concentrated luciferase solutions, which have a cytoplasmic luciferase concentration of approximately 2-4 μmol/1.     

STUDIES ON BIOLUMINESCENCE : XV. ELECTROREDUCTION OF OXYLUCIFERIN.

Oxyluciferin may be reduced to luciferin at cathodes, when an electric current is passed through the solution, or at cathodes formed by metal couples in solution, or at cathodes of oxidation-reduction cells of the NaCl - Pt - Pt - Na₂S type. It is also reduced at those metal surfaces (Al, Mn, Zn, and Cd) which liberate nascent hydrogen from water, although no visible hydrogen gas separates from the surface. Molecular hydrogen does not reduce oxyluciferin even though very finely divided but will reduce oxyluciferin in contact with palladium. Palladium has no reducing action except in presence of hydrogen, and apparently acts as a catalyst by virtue of some power of converting molecular into atomic hydrogen. Conditions are described under which a continuous luminescence of luciferin can be obtained. This luminescence may be used as a test for atomic hydrogen. It is suggested that the steady luminescence of bacteria is due to continuous oxidation of luciferin to oxyluciferin and reduction of oxyluciferin to luciferin in different parts of the bacterial cell.     

Substrate and substrate analogue binding properties of Renilla luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis catalyzes the oxidative decarboxylation of luciferin consuming 1 mol of O2 per mol of luciferin oxidized and producing 1 mol of CO2, 1 mol of oxyluciferin, and light (lambdaB, 480 nm) with a 5.5% quantum yield. In this work we have examined the binding characteristics of luciferin, luciferin analogues, and competitive inhibitors of the luciferin-luciferase reaction. The results show that luciferin binding and orientation in the single luciferin binding site of luciferase are highly specific for and dependent upon the three group substituents of the luciferin molecule while the imidazolone-pyrazine nucleus of luciferin is not directly involved in binding. Anaerobic luciferin binding promotes a rapid concentration-dependent aggregation of luciferase which results in irreversible inactivation of the enzyme. This aggregation phenomenon is not observed upon binding of oxyluciferin, luciferyl sulfate, or luciferin analogues in which the substituent at the 2 position of the imidazolone-pyrazine ring has been substantially altered.     

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin is a heat resistant enzyme, catalyzing the oxidation of coelenterazine

Blue fluorescent protein from the calcium-sensitive photoprotein aequorin (BFP-aq) was prepared and determined to be a heat resistant enzyme, catalyzing the luminescent oxidation of coelenterazine (luciferin) with molecular oxygen as a general luciferase. After treatment with excess ethylenediaminetetraacetic acid to remove Ca2+ from BFP-aq, the blue fluorescence shifted to a greenish fluorescence. This greenish fluorescent protein (gFP-aq) was identified as a non-covalent complex of apoaequorin with coelenteramide (oxyluciferin) in a molar ratio of 1:1. By incubation with coelenterazine in the absence of reducing reagents, gFP-aq was converted to aequorin at 25 degrees C. BFP-aq and gFP-aq possessing both fluorescence and luminescence activities may work as novel reporter proteins.     

Interaction of firefly luciferase with substrates and their analogs: a study using fluorescence spectroscopy methods.

The results of the author's laboratory on the interaction of Luciola mingrelica firefly luciferase with substrates and their analogs using both steady-state and time resolved fluorescence are reviewed. The contribution of fluorescence of Trp and Tyr residues of the protein to its intrinsic fluorescence spectrum was estimated. Studies of quenching of Trp and Tyr fluorescence by luciferin and ATP allowed one to determine binding constants of the luciferase with substrates and to show that the binding of one substrate to the luciferase decreases the affinity of the enzyme for the other one. Fluorescence of oxyluciferin and its analogs (dimethyl- and monomethyloxyluciferins) was shown to be a good model of native firefly bioluminescence. A comparison of the fluorescence spectra of oxyluciferin and its analogs in aqueous solutions and in the presence of the luciferase revealed specific and nonspecific effects of the microenvironment on the equilibrium between different ionic forms of oxyluciferin. An approach based on photo-physical concepts of the correlation between luminescence spectra and structure of the emitter and its microenvironment was proposed and this approach was used to analyze bioluminescence spectra of wild-type and mutant luciferases.     

Isolation of a fast decay in submillisecond Chlorella luminescence

Using a simple He-Ne (632.8-nm) laser phosphoroscope steady-state luminescence from Chlorella pyrenoidosa was studied from 50 μs to 1.1 ms between 1 ms long exciting flashes. The following results were obtained: (1) prior freezing or ultraviolet irradiation changed the time course of the luminescence to a rapid decay with a half-time of about 110 μs; (2) 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suppressed the 110-μs luminescence; (3) spectrally, all observed luminescence was, within possible error, identical to fluorescence; (4) no effect on the luminescence intensity from pulsed magnetic fields up to 30 kgauss was observed; (5) the relative fluorescence yield, measured simultaneously with luminescence, was found to be constant.Our principal conclusions, supported mainly by experiments with DCMU, are: (1) the 110-μs decay is a distinct component of the total steady-state luminescence; (2) prior freezing or ultraviolet irradiation isolates this component of the luminescence by suppressing all other components; (3) the half-time and intensity of this component are temperature independent in the interval 0–22 °C.     

Bioluminescence reaction catalyzed by membrane-bound luciferase in the “firefly squid,” Watasenia scintillans

The small Japanese “firefly squid,” Watasenia scintillans, emits a bluish luminescence from dermal photogenic organs distributed along the ventral aspects of the head, mantle, funnel, arms and eyes. The brightest light is emitted by a cluster of three tiny organs located at the tip of each of the fourth pair of arms. Studies of extracts of the arm organs show that the light is due to a luciferin-luciferase reaction in which the luciferase is membrane-bound. The other components of the reaction are coelenterazine disulfate (luciferin), ATP, Mg²⁺, and molecular oxygen. Based on the results, a reaction scheme is proposed which involves a rapid base/luciferase-catalyzed enolization of the keto group of the C-3 carbon of luciferin, followed by an adenylation of the enol group by ATP. The AMP serves as a recognition moiety for docking the substrate molecule to a luciferase bound to membrane, after which AMP is cleaved and a four-membered dioxetanone intermediate is formed by the addition of molecular oxygen. The intermediate then spontaneously decomposes to yield CO₂ and coelenteramide disulfate (oxyluciferin) in the excited state, which serves as the light emitter in the reaction.     

Control of luminescence decay and flavin binding by the LuxA carboxyl-terminal regions in chimeric bacterial luciferases.

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as P. (Vibrio) fischeri, P. phosphoreum, and P. leiognathi, have rapid decay rates. By generation of an X. luminescens-based chimeric luciferase with a 67 amino acid substitution from P. phosphoreum LuxA in the central region of the LuxA subunit, the "slow" X. luminescens luciferase was converted into a chimeric luciferase, LuxA(1)B, with a significantly more rapid decay rate. Two other chimeras with P. phosphoreum sequences substituted closer to the carboxyl terminal of LuxA, LuxA(2)B and LuxA(3)B, retained the characteristic slow decay rates of X. luminescens luciferase but had weaker interactions with both reduced and oxidized flavins, implicating the carboxyl-terminal regions in flavin binding. The dependence of the luminescence decay on concentration and type of fatty aldehyde indicated that the decay rate of "fast" luciferases arose due to a high dissociation constant (K(a)) for aldehyde (A) coupled with the rapid decay of the resultant aldehyde-free complex via a dark pathway. The decay rate of luminescence (k(T)) was related to the decanal concentration by the equation: k(T) = (k(L)A + k(D)K(a))/(K(a) + A), showing that the rate constant for luminescence decay is equal to the decay rate via the dark- (k(D)) and light-emitting (k(L)) pathways at low and high aldehyde concentrations, respectively. These results strongly implicate the central region in LuxA(1)B as critical in differentiating between "slow" and "fast" luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.     

Enhanced luciferin entry causes rapid wound-induced light emission in plants expressing high levels of luciferase

In-vivo imaging of transgenic tobacco plants (Nicotiana tobacum L.) expressing firefly luciferase under the control of the Arabidopsis phenylalanine ammonia-lyase 1 (PAL1)-promoter showed that luciferase-catalyzed light emission began immediately after the substrate luciferin was sprayed onto the leaves and reached a plateau phase after approximately 60 min. This luminescence could easily be detected for up to 24 h after luciferin application although the light intensity declined continuously during this period. A strong and rapid increase in light emission was observed within the first minutes after wounding of luciferin-sprayed leaves. However, these data did not correlate with luciferase activity analysed by an in-vitro enzyme assay. In addition, Arabidopsis plants expressing luciferase under the control of the constitutive 35S-promoter showed similar wound-induced light emission. In experiments in which only parts of the leaves were sprayed with luciferin solutions, it was shown that increased uptake of luciferin at the wound site and its transport through vascular tissue were the main reasons for the rapid burst of light produced by preformed luciferase activity. These data demonstrate that there are barriers that restrict luciferin entry into adult plants, and that luciferin availability can be a limiting factor in non-invasive luciferase assays. Received: 11 March 2000 / Accepted: 16 May 2000     

Studies in bioluminescence IV. Properties of luciferin from Pholas dactylus

Light could be obtained by the addition of Fe²⁺ to purified luciferin from Pholas dactylus in the absence of luciferase. The total light emitted was proportional to the concentration of luciferin used. The characteristics of this nonenzymic emission correspond to those of the fast reaction previously described. It may have a physiological importance since iron is present in the luciferin. The injection of Fe²⁺ alone was not sufficient; the presence of a complexing agent such as phosphate or CN⁻ or EDTA was also necessary. Light emission could also be obtained by the addition of H₂O₂, in the presence of Fe²⁺, to luciferin. It has been demonstrated that, for a given amount of luciferin, the total light emitted by the action of varying ratios of Fe²⁺ and luciferase is constant.     

Bioluminescence reaction catalyzed by membrane-bound luciferase in the "firefly squid," Watasenia scintillans

The small Japanese "firefly squid," Watasenia scintillans, emits a bluish luminescence from dermal photogenic organs distributed along the ventral aspects of the head, mantle, funnel, arms and eyes. The brightest light is emitted by a cluster of three tiny organs located at the tip of each of the fourth pair of arms. Studies of extracts of the arm organs show that the light is due to a luciferin-luciferase reaction in which the luciferase is membrane-bound. The other components of the reaction are coelenterazine disulfate (luciferin), ATP, Mg(2+), and molecular oxygen. Based on the results, a reaction scheme is proposed which involves a rapid base/luciferase-catalyzed enolization of the keto group of the C-3 carbon of luciferin, followed by an adenylation of the enol group by ATP. The AMP serves as a recognition moiety for docking the substrate molecule to a luciferase bound to membrane, after which AMP is cleaved and a four-membered dioxetanone intermediate is formed by the addition of molecular oxygen. The intermediate then spontaneously decomposes to yield CO(2) and coelenteramide disulfate (oxyluciferin) in the excited state, which serves as the light emitter in the reaction.     

Delayed fluorescence from Rhodopseudomonas sphaeroides following single flashes.

Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.     

A PRELIMINARY STUDY OF THE REDUCING INTENSITY OF LUMINOUS BACTERIA

The effect of a series of redox indicators and systems has been tested with a suspension of luminous bacteria (B. fischeri) in M/4 phosphate buffer of P_H = 7.6. The indicators behave as expected from their position in the redox series, the most positive being reduced rapidly even in presence of air and before luminescence of the bacteria disappears, those of intermediate position at the time luminescence disappears, and the more negative only long after the luminescence had ceased, due to utilization of oxygen by the bacterial respiration. Indigo monosulphonate was the only indicator not reduced on long standing of a bacterial suspension. The aerobic redox potential may be placed at an R_H = 18–20 and the anaerobic potential at an R_H = 8–10. Ferricyanides do not affect luminescence and behave as if they could not penetrate the bacterial cell. Quinone and the napthoquinones cause progressive dimming of luminescence in any concentration which affects the light but it cannot be definitely stated that this is due to rapid oxidation of luciferin although it seems likely in the case of quinone. Some indophenols dim the luminescence at first, followed by return of brightness, which is interpreted to mean rapid oxidation of luciferin while the indophenol is unreduced, more luciferin production after reduction of indophenol. The more negative redox systems do not affect the luminescence. Investigation of indicator reduction and luminescence is being continued.     

KINETICS OF THE BIOLUMINESCENT REACTION IN CYPRIDINA. II

1. The decay curve of the light produced in the course of the luminescent reaction in Cypridina is, after the first second, in complete agreement with the theoretical expectation for a monomolecular reaction, if light intensity at any instant is assumed to be proportional to reaction velocity at that instant. It is shown that for such a reaction log I = - kt + log Ak and that the experimental values satisfy this equation. 2. The first second or two of the reaction is characterized by a brilliant initial flash, whose value is much too high to accord with the succeeding intensities and with the above formula. It is suggested that this initial high reaction velocity is an indication of a heterogeneous system. 3. Identical solutions run simultaneously give decay curves which check within the limits of the photographic error. 4. Stirring does not affect the reaction velocity or the form of the decay curve. 5. Reaction velocity is proportional to enzyme concentration, over the range of concentrations used in the study. 6. Changes in the concentration of the substrate do not affect the value of k, when all other factors are held constant. A diminution of luciferin concentration results only in a decrease in the value of the y-intercept, Log Ak, the two straight line plottings for two different concentrations being parallel. 7. The temperature coefficient is high, being about 4.5 for the 15–25° interval, and 3.0 for the 25–35° interval.     

FURTHER STUDIES ON THE INHIBITION OF CYPRIDINA LUMINESCENCE BY LIGHT,WITH SOME OBSERVATIONS ON METHYLENE BLUE

1. Eosin, erythrosin, rose bengale, cyanosin, acridine, and methylene blue act photodynamically on the luminescence of a Cypridina luciferin-luciferase solution. In presence of these dyes inhibition of luminescence, which without the dye occurs only in blue-violet light, takes place in green, yellow, orange, or red light, depending on the position of the absorption bands of the dye. 2. Inhibition of Cypridina luminescence without photosensitive dye in blue-violet light, or with photosensitive dye in longer wave-lengths, does not occur in absence of oxygen. Light acts by accelerating the oxidation of luciferin without luminescence. Eosin or methylene blue act by making longer wave-lengths effective, but there is no evidence that these dyes become reduced in the process. 3. The luciferin-oxyluciferin system is similar to the methylene white-methylene blue system in many ways but not exactly similar in respect to photochemical change. Oxidation of the dye is favored in acid solution, reduction in alkaline solution. However, oxidation of luciferin is favored in all pH ranges from 4 to 10 but is much more rapid in alkaline solution, either in light or darkness. There is no evidence that reduction of oxyluciferin is favored in alkaline solution. Clark''s observation that oxidation (blueing) of methylene white occurs in complete absence of oxygen has been confirmed for acid solutions. I observed no blueing in light in alkaline solution.     

Biochemical and morphological changes accompanying light organ development in the firefly, Photuris pennsylvanica

The larval light organs of the firefly, Photuris pennsylvanica, regress and are replaced by the adult lantern during metamorphosis. Larval and adult light organs are present and capable of periodic light emission during the latter stages of pupation and the early adult. The whole pupa emits a continuous, low level, glow throughout pupation.During pupation levels of luciferase and luciferin, the enzyme and substrate required in the light reaction, were found to remain constant in the posterior half of the pupa and to show an initial increase followed by a decrease in the anterior half. Levels of luciferase and luciferin in anterior halves were not affected by ablation of the larval light organs. The ratio of luciferase to luciferin concentrations changed from less than 1, in larval and pupal stages, to greater than 1, in the adult. Changes in the concentration and the localization of luciferase and luciferin were correlated with observed light organ development.     

Use of Firefly Luciferase for ATP Measurement: Other Nucleotides Enhance Turnover

Firefly luciferase utilizes only ATP and a few closely related nucleotides as substrates for the formation of luciferyl adenylate which is an intermediate in the bioluminescent reaction sequence that oxidizes firefly luciferin. The enzyme shows two different time courses of light production depending on ATP concentration used: a flash with high concentrations of ATP (>8μM) or a fairly constant production of light with lower concentrations of ATP (< 1 μM). Many nucleotides, nucleotide-containing substances and other compounds, when added either prior to or 1 min after the addition of ATP, change the time course of light production. When added before ATP, these compounds yield a reaction mixture in which light production is fairly constant (at the level characteristic of the flash observed with that ATP concentration). When the compounds are added after ATP addition, light production is markedly stimulated and the higher rate of light production is maintained for several minutes. There is an increase in quanta of light produced per luciferase dimer from 1 to 5/min with the addition of any of several nucleotide analogues. These results are consistent with a stimulated release of the inhibitory product oxyluciferin, allowing turnover of the enzyme. This enzyme turnover permits more light output at high ATP concentrations, thus enhancing the sensitivity of enzyme determination.