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Advances in the knowledge of light emission by firefly luciferin and oxyluciferin
Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Short Review
Advances in the knowledge of light emission by firefly luciferin and oxyluciferin João Vieira, Luís Pinto da Silva, Joaquim C.G. Esteves da Silva ⇑ Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 19 April 2012 Received in revised form 20 August 2012 Accepted 27 August 2012 Available online 5 September 2012 Keywords: Bioluminescence Luciferase Fluorescence Oxyluciferin Luciferin Firefly
a b s t r a c t Firefly luciferase is the most important and studied bioluminescence system. Due to very interesting characteristics, this system has gained numerous biomedical, pharmaceutical and bioanalytical applications, among others. In order to improve the use of this system, various researchers have tried to understand experimentally the colour of bioluminescence, and to create ways of tuning the colour emitted. The objective of this manuscript is to review the experimental studies of firefly luciferin and oxyluciferin, and related analogues, fluorescence and bioluminescence. Ó 2012 Elsevier B.V. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-LH2 and analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. 2.2. Amino analogues of D-LH2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. OxyLH2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. OxyLH2 analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioluminescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Bioluminescence emission by OxyLH2 analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Bioluminescence emission by point mutated luciferase enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Bioluminescence is considered to be a light emission phenomenon, which originates from an enzyme-catalysed chemical reaction. The most studied bioluminescence system is that of the fireflies. Firefly luciferase catalyses a two-step reaction: firefly luciferin (D-LH2, Scheme 1) reacts with adenosine-50 -triphosphate (ATP), subsequently generating luciferyl-adenylate (LH2-AMP); The latter molecule will then be oxidised, in the presence of molec⇑ Corresponding author. Tel.: +351 226082869; fax: +351 226082959. E-mail addresses: [email protected] (J. Vieira), [email protected] (L. Pinto da Silva), [email protected] (J.C.G. Esteves da Silva). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.08.017
33 35 35 36 37 37 37 37 37 38 38 38
ular oxygen, which results in the formation of oxyluciferin (OxyLH2), CO2 and adenosine-50 -monophosphate (AMP) [1–4]. OxyLH2 is usually thought to exist in solution as one of six possible species, due to a complex chemical equilibrium (Scheme 2). However, it is known that in the bioluminescence reaction, this molecule is formed in its anionic keto-form (Keto-(-1), Scheme 2) [5–7]. Keto-(-1) is formed in the excited state, due to the formation and subsequent decomposition of firefly dioxetanone [8,9]. The bioluminophore will then decay to the ground state, with emission of green light [4,10–12]. The colour of bioluminescence was demonstrated to be modulated by intermolecular interactions (mostly electrostatic, p–p stacking and hydrogen-bonding) and the polarity of the microenvironment [5,7,13–20].
34
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39 Table 2 Emission spectra of D-LH2. Compound ( O)-LH2
Ethanol (0.654) Methanol (0.762) Ethanol (0.654) Ethanolb (0.654) Ethanola (0.654) Acetonitrile (0.460) Methanol (0.762) Ethanol (0.654) Ethanolb (0.654) p-Dioxanea (0.164)
(HO)-LH2
a
Scheme 1.
D-LH2
b
and analogues.
Due to very interesting characteristics, the firefly bioluminescence system has been gaining numerous biomedical, pharmaceutical, bioanalytical and bioimaging applications, among others [21–26]. The modulation of the colour of bioluminescence would be of extreme importance for a better use of this system. Red-emitting luciferase-Keto-(-1) complexes could be used in in vivo medical imaging, as red light is absorbed very poorly by mammalian tissues in comparison with the natural colour of the emitted light. Furthermore, it was hypothesised that the control of the colour of bioluminescence could be the basis for using luciferase as a single
Solvent (ET (kcal mol1)) a
kem (nm)
Refs.
532 530 530 450 444 430 425 425 404 399
[33] [34] [34] [33] [33] [35] [34] [34] [33] [33]
Measured at room temperature. Measured at 77 K.
dual reporter gene, a bioindicator of cellular stress, and a probe for intracellular changes of pH [3]. The objective of this manuscript is to review the experimental findings regarding the fluorescence and bioluminescence of LH2 and OxyLH2, and related analogues, in several conditions of polarity, pH and in different luciferase enzymes. With this review, we intend to summarise the knowledge obtained so far, regarding the effect of structural and environmental effects on the colour of light emitted by these molecules.
Scheme 2. Possible forms of OxyLH2 in solution.
Table 1 Absorption and emission spectra of D-LH2 and analogues. Compound
Solvent (ET (kcal mol1))
pH
kabs (nm)
kem (nm) (quantum yield)
Refs.
(HO)-LH2
Tris–acetate buffera Water (1.000) Neutral aqueous solution Ethanol > 99%, acetate buffer HCl 20–220 mM Water (1.000) Ethanol 90%, carbonate buffer Water (1.000) Ethanol 98%, acetate buffer Water (1.000) Water (1.000) Neutral aqueous solution Tris–acetate buffera Ethylene glycol 95%, acetate buffer Water (1.000) Tris–acetate buffera Water (1.000) Water (1.000) Ethanol 99%, acetate buffer Water (1.000)
7.7 5.0
330 328 330 331 396 348 336 – – 384 – 350 – 354 327 323 323 325 407
530 (0.90) 542 (0.25) 440 435 (0.03) 590 560 (0.62) 560 (0.26) 551 (0.11) 550 (0.62) 435 (0.20) 542 (0.62) 530 520 457 (0.25) (0.01) 450 441 (0.03b) 441 (0.03b) 420 (0.01b) (0.01)
[30] [27] [35] [27] [36] [27] [27] [27] [27] [27] [27] [35] [30] [27] [27] [30] [27] [27] [27] [27]
+HNRO (O)-L (HO)-LH2-AMP (HO)-L (O)-LH2 AL (HO)-L-AMP Gly–AL MeOLH2
(O)-L-AMP a b
1 mM EDTA, 0.2 M NaCl. Approximate value owing to extreme photosensitivity.
35
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39
Scheme 3. L and adenylate.
2. Fluorescence 2.1. D-LH2 and analogues The data referring to the absorption and emission of D-LH2 and analogues are presented in Tables 1 and 2. The structures of these molecules are schematically represented in Schemes 1 and 3. It has been documented that a redshift in the emission wavelength of D-LH2 occurs by increasing the temperature or lowering the pH [27–32]. A dependence on solvent polarity has also been proposed, as luciferin fluorescence at neutral pH is known to be
blue in solvents which prevent proton transfer and/or of medium polarity, such as DMSO, methanol, ice or acetone, and yellow– green in water [27]. An early study by Jung et al. [33], in which the light emitted by the phenolate form (O)-LH2 (Scheme 1) in ethanol shifted from 450 nm at 77 K to 532 nm at room temperature, backs up the correlation between temperature and fluorescence. In the same study, a similar shift was observed for the neutral (HO)-LH2 form (Scheme 1). On the other hand, Morton et al. [27] demonstrated that for the analogue 60 -methoxyLH2 (MeOLH2, Scheme 1), while the emission spectrum suggested sensitivity to solvent polarity, there was no observable variation in colour at different pH values. The authors noted, however, that the reported values were approximated due to the extreme photosensitivity of the compound, and therefore not entirely reliable. Three separate experiments [33–35] produced concordant results regarding the difference in emission wavelength between the neutral and the phenolate forms of LH2. In different mediums, the phenolate form emitted closer to red than the neutral form. This deviation seems to carry over to some luciferin analogues, namely dehydroLH2 (L, Scheme 3) and its adenylated form (LAMP, Scheme 3) [27]. In both cases, either the absorption or emis-
Table 3 Absorption and emission spectra of amino analogues of D-LH2 [37]. Solvent (ET (kcal mol1))a
NaPi Methanol (0.762) Acetonitrile (0.460) DMSO (0.444) Dimethylformamide (0.386) Dichloromethane (0.309) Chloroform (0.259) THF (0.207) Benzene (0.111) a
kabs (nm)
kem (nm)
AL
QAL
NAL
CAL
AL
QAL
NAL
CAL
349 363 362 377 363 357 357 367 356
353 363 364 380 372 361 359 372 358
322 327 333 342 329 332 329 334 327
399 414 401 415 408 393 393 403 396
523 492 478 487 479 463 466 461 437
500 479 460 468 464 438 439 438 423
465 440 425 437 425 410 412 417 400
458 461 462 472 468 461 457 462 469
Measurements at pH 7.4 in NaPi and pH 7.7 in remaining solvents.
Scheme 4. Amino analogues of D-LH2.
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J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39
Table 4 Absorption and emission spectra of OxyLH2. Solvent (ET (kcal mol1))
kabs (nm)
kem (nm) (quantum yield)
Refs.
Water (1.000) DMSO, excess PhOK Chloroform (0.259) Dichloromethane (0.309) Acetonitrile (0.460) Methanol (0.762) DMSO (0.444) Acetone (0.355)
409 475 376 369 369 372 377 367
553 570 471 465 457 457 448 436
[38] [28] [38] [38] [38] [38] [38] [38]
(0.470) (0.308) (0.231) (0.305) (0.345) (0.511) (0.277)
sion spectra of the phenolate form peaked at longer wavelengths than its protonated counterpart. In a series of studies by Erez et al. [34–36], an emission peak at approximately 590 nm in acidic aqueous solution was attributed to the zwitterion form of D-LH2 (+HNRO-), which predominated over the phenolate and neutral forms’ emission bands at 220 mM HCl [36]. Adenylation of LH2 produced a redshift in both the absorption and emission spectra in water [27]. Although L-AMP also displayed
a peak in absorption closer to red, no conclusion could be drawn in regards to its emission on account of the low quantum yield of the experiment. 2.2. Amino analogues of D-LH2 The data referring to the absorption and emission of aminoLH2 (AL) and analogues are presented in Table 3, while their structures are schematically represented in Scheme 4. A study by Nagano et al. [37] showed that the fluorescence spectrum of coumarylAL (CAL), an analogue of LH2 bearing an amino group, is practically independent of solvent polarity, whereas in general those of AL, naphtylAL (NAL) and quinolylAL (QAL) shift to red in more polar solvents, even though there is no discernible pattern when solvents of medium polarity are taken into account. CAL also differs from the other amino analogues in that it displays fluorescence at lower wavelengths than D-LH2. An emission peak at 450 nm in buffer was reported for glycineD-AL (Gly–AL) [30]. The authors proposed that the lower ionisation level of the glycine group could be responsible for the low fluores-
Table 5 Absorption and emission spectra of OxyLH2 analogues. Compound
kem (nm) (quantum yield)
Refs.
Tris–acetate buffer DMSO (0.444) Methanol (0.762) 2-Propanol (0.546) Acetonitrile (0.460) Chloroform (0.259) Benzene (0.111) p-Xylene (0.074)
485 – – – – – – –
639 630 630 624 624 588 541 541
[40] [32] [32] [32] [32] [32] [32] [32]
DMOxyLH2
Water (1.000) p-Xylene (0.074) Benzene (0.111) DMSO, excess PhOK Methanol (0.762) DMSO (0.444) 2-Propanol (0.546) Acetonitrile (0.460) Chloroform (0.259)
486 366 366 580 388 579 526 372 370
640c (0.04) 420 (0.001) 420 (0.001) 633 (0.62) 526 (0.19) 522 (0.07) 504 (0.11) 479 (0.03) 454 (0.001)
MOxyLH2 dianion
Tris–acetate buffera
440
550
DHOxyLH2
Water (1.000) DMSO (0.444) Dimethylformamide (0.386) 1-Butanol (0.586) Ethanol (0.654) Methanol (0.762) 2-Propanol (0.546) CCl4 (0.052) Hexane (0.009) Toluene (0.099) Benzene (0.111) 1,2-Dichloroethane (0.327) m-Xylene Acetone (0.355) Acetonitrile (0.460) Dichloromethane (0.309) Chloroform (0.259) Ethyl acetate (0.228)
361 367 365 363 362 361 361 367 360 366 366 359 367 358 355 348 356 359
541 451 449 441 440 440 439 439 439 439 438 436 435 434 434 434 433 432
(0.156) (0.185) (0.212) (0.42) (0.242) (0.073) (0.262) (0.676) (0.283) (0.711) (0.767) (0.610) (0.601) (0.51) (0.883) (0.40) (0.279) (0.544)
[39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39]
MDMOxyLH2
p-Xylene (0.074) Water (1.000) Methanol (0.762) DMSO (0.444) 2-Propanol (0.546) Acetonitrile (0.460) Chloroform (0.259) Benzene (0.111)
368 380b 378 380 380 373 380 371
Weak 535 505 (0.12) 499 (0.008) 487 (0.02) 478 (0.02) 454 (0.002) 440 (0.001)
[32] [32] [32] [32] [32] [32] [32] [32]
( O)-DMOxyLH2
a b c
Solvent (ET (kcal mol1))
kabs (nm)
a
2 mM EDTA, 10 mM MgSO4, 1 mM dithiothreitol. Not determined accurately due to decomposition of the substrate. Fluorescence attributed to the phenolate ion.
(0.53) (0.08) (0.29) (0.26) (0.75) (0.82) (0.46)
[32] [32] [32] [28] [32] [32] [32] [32] [32] [40]
37
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39 Table 6 Bioluminescence of D-LH2, OxyLH2 and analogues. Compound
Enzyme
Bioluminescence (nm)
Refs.
D-LH2
Luc
560
[30]
AL CAL
Luc Luc Luc
596 500 578
[37] [37] [30]
Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc CBRLuc CBRLuc CBRLuc CBRLuc PplGR Ppy Ppy, Luc, PplGR
615 526 564 601 556 587 570 618 615 515 560 588 559 570 450 582 499 547 548 624 560 No emission
[37] [37] [37] [37] [40] [40] [30] [40] [37] [37] [37] [37] [37] [37] [37] [37] [37] [37] [37] [41] [41] [41]
D-AL
Scheme 5. OxyLH2 analogues.
cence peak, tying the result to earlier experiments by another group on MeOLH2 and analogues. 2.3. OxyLH2 The data referring to the absorption and emission of OxyLH2 are presented in Table 4, while their structures are schematically represented in Scheme 2. As with D-LH2 and some of its analogues, the phenolate ion of OxyLH2 emits closer to red than its protonated form [32,38]. Spectroscopic studies with an ample range of solvents showed that there is no linearity between solvent polarity and emission wavelength, possibly due to the presence of several forms of OxyLH2 in solution [32,38]. In an article regarding the spectral–structural effect of the equilibria between these forms, Naumov and Kochunnoonny stated that if only the enol-enolate-keto equilibrium is taken into account, greater solvent polarity does not necessarily translate into a greater redshift in luminescence [39]. 2.4. OxyLH2 analogues The data referring to the absorption and emission of OxyLH2 analogues are presented in Table 5, while their structures are schematically represented in Scheme 5. Substitution of both hydrogen atoms in the thiazolone moiety for methyl groups shifted the emission peak towards greater wavelengths, with the phenolate form emitting closer to red than the neutral form. Further substitution of the hydroxyl in position 60 for a methoxy group caused a small blueshift [32] for 60 -methoxy-5,5-dimethylOxyLH2 (MDMOxyLH2), parallel to the effect observed for MeOLH2 [27]. This analogue showed decomposition in water, presumably due to hydrolysis of the thiazolone ring. MDMOxyLH2 and 5,5-dimethylOxyLH2 (DMOxyLH2) absorbed at around the same wavelengths in solvents of different polarity and showed considerable diversity in emission maxima. Additionally, both compounds presented very low quantum yields in less polar solvents. By comparing their emission spectra with that of bioluminescence, the authors ruled out the neutral form of OxyLH2 as the light-emitting species [32]. Ugarova proposed that while 5-methylOxyLH2 (MOxyLH2), like OxyLH2, can exist in six forms, DMOxyLH2 can only be found in its phenolate and 60 -OH forms, as the steric hindrances caused by the two methyl groups obstruct any significant changes in the adjacent C–O double bond [40]. DehydroxyOxyLH2 (DHOxyLH2) emitted at 541 nm in water and produced mostly violet light (432–451 nm) in several solvents of lower polarity. The authors attributed these results to the role of the hydroxyl anion in lowering the energy of the excited state at physiological pH, which would partially account for a redshift of the emitted light; in the absence of this group, and as a result of
DMAL DMCAL DMNAL DMQAL Enolate-OxyLH2 Enol-OxyLH2 Gly–AL Keto-OxyLH2 MAL MCAL MNAL MQAL NAL QAL MOxyLH2 dianion AL CAL NAL QAL D-DMLH2-AMP D-DMLH2
inefficient charge transfer, DHOxyLH2 can only emit orange light [39].
3. Bioluminescence 3.1. Bioluminescence emission by OxyLH2 analogues The data referring to bioluminescence emission by OxyLH2 analogues is presented in Table 6. No bioluminescence was observed for 5,5-dimethylLH2 (DMLH2). It was suggested that the addition of the two methyl groups interferes with the orientation of the carboxylate ion, ultimately preventing the adenylation of the compound due to steric hindrances. Spectroscopic studies of its adenylated form (DMLH2AMP) show that these hindrances may also have a negative influence in the oxidation step of the reaction [41]. Analysis of the Ppy- and PplGR-catalysed reactions involving D-DMLH2-AMP, of which DMOxyLH2 was the resulting compound, led the authors to conclude that red and green bioluminescence could be attributed to the keto form of this product [41]. Methylation of NAL was shown to cause a minor variation in its bioluminescence spectrum. Methyl- and dimethyl-forms of AL, QAL and CAL absorbed and emitted light at greater wavelengths [37]. 3.2. Bioluminescence emission by point mutated luciferase enzymes The data referring to bioluminescence emission by point mutated luciferase enzymes is presented in Table 7. Point mutations of residues His245, Arg215, Thr343 or Arg337 were shown to change the emission peak of Photinus pyralis luciferase towards the red region. The authors noted that there was no correlation between polarizability and emission wavelength for His245 mutants, and that the consequent spectral changes can possible be explained by a combination of effects of the protein microenvironment as a whole rather than by particular properties of residues that were introduced, pointing out that native muta-
38
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39 Table 7 Bioluminescence of wild-type and mutated luciferases [12].
For their prospective applications as substrates in bioluminescence assays, analogues of D-LH2 and OxyLH2 were analysed in regards to their spectroscopic properties under enzyme-catalysed reaction with various luciferases (most notably Luc and Ppy), and while these studies were not focused or conclusive in the search for correlations between light emission and external factors (either environmental or compound-related), they may also benefit from any significant discoveries on this subject, advancing the enhancement of the Luc system in its current medical and analytical uses. There has also been an attempt to understand the individual role of certain residues in the active site of firefly luciferase and in what capacity a modification in those positions would affect the bioluminescence reaction. Synthetic and natural mutants of luciferases effectively produced a wider range of colours than the respective wild types, and a redshifting effect was attributed to the insertion of positive residues in the active site. Nevertheless, there are no definitive answers concerning a specific position that stands out from the others, and it has been noted that the modification of residues further away from the active site is not inconsequential, which adds to the difficulty of narrowing down the search for key regions in the enzyme. Such results emphasise the current assumption that the research on the overall mechanism of colour modulation must not take the possible factors into account separately, but rather encompass all the variables that are known to influence the outcome of the light-emitting process and eventually uncover any connections between them.
Enzyme (inset: mutation)
Bioluminescence (nm)
Luciola mingrelica Ser286Lys Ser286Gln Ser286Tyr Ser286Leu His433Tyr
570 608 609 613 619 606
Luciola cruciata Ser286Asn Gly326Ser His433Tyr Pro452Ser
562 607 609 612 595
Photinus pyralis Native His245Phe His245Ala His245Arg His245Gln His245Asn His245Asp Thr343Ser Thr343Ala Arg218Lys Arg218Gln Arg218Ala Arg337Lys Arg337Gln
562 558 595 604 579 606 613 617 560 617 572 608 611 595 594
Hotaria parvula His433Tyr
568 610
Acknowledgments
Phrixothrix viviani Arg215Ser
549 589
Financial support from Fundação para a Ciência e Tecnologia (FCT, Lisbon) (Programa Operacional Temático Factores de Competitividade (COMPETE) e comparticipado pelo Fundo Comunitário Europeu (FEDER) (Project PTDC/QUI/71366/2006) is acknowledged. A Ph.D. Grant to Luís Pinto da Silva (SFRHn76612n2011), attributed by FCT, is also acknowledged.
tions of residues distant from the active site also produced redshift [12]. Mutations of Ser286 for amino acids which increased the orientation polarizability in that position were responsible for redshifts in Luciola cruciata and Luciola mingrelica luciferases [12]. Substitution of the hystidine residue in position 433 for a tyrosine caused a redshift in both enzymes [12] and produced the same result in that of Hotaria parvula [40].
4. Conclusion The effect of variations in pH and temperature on the emitted light has been confirmed in several instances and is uncontroversial in respect to D-LH2. It is known that either a decrease in pH or an increase in temperature leads to a redshift in its fluorescence wavelength. Additionally, consistent evidence pointing to a difference between the emission spectra of the neutral ligands and those of their respective phenolate ions, with the latter forms tending to emit light closer to red, further support the role of pH in colour modulation. It has been established, however, that these factors alone would not control the light-emitting process in its entirety. As other studies with analogues of D-LH2 and OxyLH2 indicated that they were affected differently by pH and temperature changes, it became clear that other aspects of the system had to be taken into account. Spectroscopic studies of LH2-related compounds in various mediums were carried out in an effort to correlate emission wavelength and solvent polarity, and despite the lack of linearity between the two, it has been proven that in general more polar solvents (i.e. water and methanol) cause a redshift compared to mediums of very low polarity.
References [1] S.M. Marques, J.C. Esteves da Silva, Firefly bioluminescence. a mechanistic approach of luciferase catalyzed reactions, IUBMB Life 61 (1) (2009) 6–17. [2] J.M. Leitão, J.C. Esteves da Silva, Firefly luciferase inhibition, J. Photochem. Photobiol. B. 101 (1) (2010) 1–8. [3] V.R. Viviani, F.G. Arnoldi, A.J. Neto, T.L. Oehlmeyer, E.J. Bechara, Y. Ohmiya, The structural origin and biological function of pH-sensitivity in firefly luciferases, Photochem. Photobiol. Sci. 7 (2) (2008) 159–169. [4] L.P. da Silva, J.C. Esteves da Silva, Computational studies of the luciferase lightemitting product: oxyluciferin, J. Chem. Theory Comput. 7 (4) (2011) 809–817. [5] L.P. da Silva, J.C. Esteves da Silva, Computational investigation of the effect of pH on the color of firefly bioluminescence by DFT, ChemPhysChem 12 (5) (2011) 951–960. [6] S.F. Chen, Y.J. Liu, I. Navizet, N. Ferre, W.H. Fang, R. Lind, Systematic theoretical investigation on the light emitter of firefly, J. Chem. Theory Comput. 7 (3) (2011) 798–803. [7] C.I. Song, Y.M. Rhee, Dynamics on the electronically excited state surface of the bioluminescent firefly luciferase-oxyluciferin system, J. Am. Chem. Soc. 133 (31) (2011) 12040–12049. [8] M. Matsumoto, Advanced chemistry of dioxetane-based chemiluminescent substrates originating from bioluminescence, J. Photochem. Photobiol. C. 5 (1) (2004) 27–53. [9] L. Pinto da Silva, J.C.G. Esteves da Silva, Firefly chemiluminescence and bioluminescence. Efficient generation of excited states, ChemPhysChem 13 (9) (2012) 2257–2262. [10] J.Y. Hasegawa, K.J. Fujimoto, H. Nakatsuji, Color tuning in photofunctional proteins, ChemPhysChem 12 (17) (2011) 3106–3115. [11] S. Hosseinkhani, Molecular enigma of multicolor bioluminescence of firefly luciferase, Cell Mol. Life Sci. 68 (7) (2011) 1167–1182. [12] N.N. Ugarova, L.Y. Brovko, Protein structure and bioluminescent spectra for firefly bioluminescence, Luminescence 17 (5) (2002) 321–330. [13] L.P. da Silva, J.C. Esteves da Silva, Theoretical modulation of the color of light emitted by firefly oxyluciferin, J. Comput. Chem. 32 (12) (2011) 2654–2663. [14] L.P. da Silva, J.C. Esteves da Silva, Study on the effects of intermolecular interactions on firefly multicolor bioluminescence, ChemPhysChem 12 (16) (2011) 3002–3008.
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39 [15] L.P. da Silva, J.C. Esteves da Silva, TD-DFT/molecular mechanics study of the Photinus pyralis bioluminescence system, J. Phys. Chem. B. 116 (6) (2012) 2008–2013. [16] L.P. da Silva, J.C. Esteves da Silva, Theoretical analysis of the color tuning mechanism of oxyluciferin and 5-hydroxyoxyluciferin, Comput. Theor. Chem. 988 (2012) 56–62. [17] A. Tagami, N. Ishibashi, D. Kato, N. Taguchi, Y. Mochizuki, H. Watanabe, M. Ito, S. Tanaka, Ab initio quantum-chemical study on emission spectra of bioluminescent luciferases by fragment molecular orbital method, Chem. Phys. Lett. 472 (1–3) (2009) 118–123. [18] N. Nakatani, J.Y. Hasegawa, H. Nakatsuji, Red light in chemiluminescence and yellow–green light in bioluminescence. Color-tuning mechanism of firefly, Photinus pyralis, studied by the symmetry-adapted cluster-configuration interaction method, J. Am. Chem. Soc. 129 (28) (2007) 8756–8765. [19] B.R. Branchini, T.L. Southworth, M.H. Murtiashaw, R.A. Magyar, S.A. Gonzalez, M.C. Ruggiero, J.G. Stroh, An alternative mechanism of bioluminescence color determination in firefly luciferase, Biochemistry 43 (23) (2004) 7255–7262. [20] C.G. Min, A.M. Ren, J.F. Guo, Z.W. Li, L.Y. Zou, J.D. Goddard, J.K. Feng, A timedependent density functional theory investigation on the origin of red chemiluminescence, ChemPhysChem 11 (1) (2010) 251–259. [21] A. Roda, M. Guardigli, Analytical chemiluminescence and bioluminescence. Latest achievements and new horizons, Anal. Bioanal. Chem. 402 (1) (2012) 69–76. [22] L. Mezzanotte, I. Que, E. Kaijzel, B.R. Branchini, A. Roda, C. Löwik, Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase, PLoS ONE 6 (4) (2011) e1. 9277. [23] B.R. Branchini, E. Michelini, L. Cenenini, A. Roda, A portable bioluminescence engineered cell-based biosensor for on-site applications, Biosens. Bioelectron. 26 (8) (2011) 3647–3653. [24] S.M. Marques, F. Peralta, J.C. Esteves da Silva, Optimized chromatographic and bioluminescent methods for inorganic pyrophosphate based on its conversion to ATP by firefly luciferase, Talanta 77 (4) (2009) 1497–1503. [25] S.M. Marques, J.C. Esteves da Silva, An optimized luciferase bioluminescent assay for coenzyme A, Anal. Bioanal. Chem. 391 (6) (2008) 2161–2168. [26] E. Michelini, L. Cenenini, L. Mezzanotte, A. Roda, Luminescent probes and visualization of bioluminescence, Methods Mol. Biol. 574 (2009) 1–13. [27] R.A. Morton, T.A. Hopkins, H.H. Seliger, Spectroscopic properties of firefly luciferin and related compounds; an approach to product emission, Biochemistry 8 (4) (1969) 1598–1607.
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[28] E.H. White, M.G. Steinmetz, J.D. Miano, P.D. Wildes, R. Morland, Chemi- and bioluminescence of firefly luciferin, J. Am. Chem. Soc. 102 (9) (1980) 3199– 3208. [29] V.R. Viviani, The origin, diversity, and structure function relationships of insect luciferases, Cell. Mol. Life Sci. 59 (2002) 1833–1850. [30] R. Shinde, J. Perkins, C.H. Contag, Luciferin derivatives for enhanced in vitro and in vivo bioluminescence assays, Biochemistry 45 (37) (2006) 11103–11112. [31] V.R. Viviani, F.G.C. Arnoldi, A.J.S. Neto, T.L. Oehlmeyer, E.J.H. Bechara, Y. Ohmiya, The structural origin and biological function of pH-sensitivity in firefly luciferases, Photochem. Photobiol. Sci. 7 (2008) 159–169. [32] T. Hirano, Y. Hasumi, K. Ohtsuka, S. Maki, H. Niwa, M. Yamaji, D. Hashizume, Spectroscopic studies of the light-color modulation mechanism of firefly (beetle) bioluminescence, J. Am. Chem. Soc. 131 (2009) 2385–2396. [33] J. Jung, C. Chin, P. Song, Electronic excited states of D-(-)-luciferin and related chromophores, J. Am. Chem. Soc. 98 (13) (1976) 3949–3954. [34] I. Presiado, Y. Erez, D. Huppert, Excited-state intermolecular proton transfer of the firefly’s chromophore D-luciferin. 2. Watermethanol mixtures, J. Phys. Chem. A. 114 (35) (2010) 9471–9479. [35] Y. Erez, D. Huppert, Excited-state intermolecular proton transfer of the firefly’s chromophore D-luciferin, J. Phys. Chem. A. 114 (31) (2010) 8075–8082. [36] Y. Erez, I. Presiado, R. Gepshtein, D. Huppert, Excited-state intermolecular proton transfer of firefly luciferin IV. Temperature and pH dependence, J. Phys. Chem. A. 115 (9) (2011) 1617–1626. [37] H. Takakura, K. Sasakura, T. Ueno, Y. Urano, T. Terai, K. Hahaoka, T. Tsuboi, T. Nagano, Development of luciferin analogues bearing an amino group and their application as BRET donors, Chem. – An Asial J. 5 (2010) 2053–2061. [38] P. Naumov, Y. Ozawa, K. Ohkubo, S. Fukuzumi, Structure and spectroscopy of oxyluciferin, the light emitter of the firefly bioluminescence, J. Am. Chem. Soc. 131 (32) (2009) 11590–11605. [39] P. Naumov, M. Kochunnoonny, Spectralstructural effects of the ketoenolenolate and phenolphenolate equilibria of oxyluciferin, J. Am. Chem. Soc. 132 (33) (2010) 11566–11579. [40] N.N. Ugarova, Interaction of firefly luciferase with substrates and their analogs: a study using fluorescence spectroscopy methods, Photochem. Photobiol. Sci. 7 (2008) 218–227. [41] B.R. Branchini, M.H. Murtiashaw, R.A. Magyar, N.C. Portier, M.C. Ruggiero, J.G. Stroh, Yellow–green and red firefly bioluminescence from 5,5dimethyloxyluciferin, J. Am. Chem. Soc. 124 (10) (2002) 2112–2113.
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Short Review
Advances in the knowledge of light emission by firefly luciferin and oxyluciferin João Vieira, Luís Pinto da Silva, Joaquim C.G. Esteves da Silva ⇑ Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal
a r t i c l e
i n f o
Article history: Received 19 April 2012 Received in revised form 20 August 2012 Accepted 27 August 2012 Available online 5 September 2012 Keywords: Bioluminescence Luciferase Fluorescence Oxyluciferin Luciferin Firefly
a b s t r a c t Firefly luciferase is the most important and studied bioluminescence system. Due to very interesting characteristics, this system has gained numerous biomedical, pharmaceutical and bioanalytical applications, among others. In order to improve the use of this system, various researchers have tried to understand experimentally the colour of bioluminescence, and to create ways of tuning the colour emitted. The objective of this manuscript is to review the experimental studies of firefly luciferin and oxyluciferin, and related analogues, fluorescence and bioluminescence. Ó 2012 Elsevier B.V. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-LH2 and analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. 2.2. Amino analogues of D-LH2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. OxyLH2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. OxyLH2 analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bioluminescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Bioluminescence emission by OxyLH2 analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Bioluminescence emission by point mutated luciferase enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Bioluminescence is considered to be a light emission phenomenon, which originates from an enzyme-catalysed chemical reaction. The most studied bioluminescence system is that of the fireflies. Firefly luciferase catalyses a two-step reaction: firefly luciferin (D-LH2, Scheme 1) reacts with adenosine-50 -triphosphate (ATP), subsequently generating luciferyl-adenylate (LH2-AMP); The latter molecule will then be oxidised, in the presence of molec⇑ Corresponding author. Tel.: +351 226082869; fax: +351 226082959. E-mail addresses: [email protected] (J. Vieira), [email protected] (L. Pinto da Silva), [email protected] (J.C.G. Esteves da Silva). 1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2012.08.017
33 35 35 36 37 37 37 37 37 38 38 38
ular oxygen, which results in the formation of oxyluciferin (OxyLH2), CO2 and adenosine-50 -monophosphate (AMP) [1–4]. OxyLH2 is usually thought to exist in solution as one of six possible species, due to a complex chemical equilibrium (Scheme 2). However, it is known that in the bioluminescence reaction, this molecule is formed in its anionic keto-form (Keto-(-1), Scheme 2) [5–7]. Keto-(-1) is formed in the excited state, due to the formation and subsequent decomposition of firefly dioxetanone [8,9]. The bioluminophore will then decay to the ground state, with emission of green light [4,10–12]. The colour of bioluminescence was demonstrated to be modulated by intermolecular interactions (mostly electrostatic, p–p stacking and hydrogen-bonding) and the polarity of the microenvironment [5,7,13–20].
34
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39 Table 2 Emission spectra of D-LH2. Compound ( O)-LH2
Ethanol (0.654) Methanol (0.762) Ethanol (0.654) Ethanolb (0.654) Ethanola (0.654) Acetonitrile (0.460) Methanol (0.762) Ethanol (0.654) Ethanolb (0.654) p-Dioxanea (0.164)
(HO)-LH2
a
Scheme 1.
D-LH2
b
and analogues.
Due to very interesting characteristics, the firefly bioluminescence system has been gaining numerous biomedical, pharmaceutical, bioanalytical and bioimaging applications, among others [21–26]. The modulation of the colour of bioluminescence would be of extreme importance for a better use of this system. Red-emitting luciferase-Keto-(-1) complexes could be used in in vivo medical imaging, as red light is absorbed very poorly by mammalian tissues in comparison with the natural colour of the emitted light. Furthermore, it was hypothesised that the control of the colour of bioluminescence could be the basis for using luciferase as a single
Solvent (ET (kcal mol1)) a
kem (nm)
Refs.
532 530 530 450 444 430 425 425 404 399
[33] [34] [34] [33] [33] [35] [34] [34] [33] [33]
Measured at room temperature. Measured at 77 K.
dual reporter gene, a bioindicator of cellular stress, and a probe for intracellular changes of pH [3]. The objective of this manuscript is to review the experimental findings regarding the fluorescence and bioluminescence of LH2 and OxyLH2, and related analogues, in several conditions of polarity, pH and in different luciferase enzymes. With this review, we intend to summarise the knowledge obtained so far, regarding the effect of structural and environmental effects on the colour of light emitted by these molecules.
Scheme 2. Possible forms of OxyLH2 in solution.
Table 1 Absorption and emission spectra of D-LH2 and analogues. Compound
Solvent (ET (kcal mol1))
pH
kabs (nm)
kem (nm) (quantum yield)
Refs.
(HO)-LH2
Tris–acetate buffera Water (1.000) Neutral aqueous solution Ethanol > 99%, acetate buffer HCl 20–220 mM Water (1.000) Ethanol 90%, carbonate buffer Water (1.000) Ethanol 98%, acetate buffer Water (1.000) Water (1.000) Neutral aqueous solution Tris–acetate buffera Ethylene glycol 95%, acetate buffer Water (1.000) Tris–acetate buffera Water (1.000) Water (1.000) Ethanol 99%, acetate buffer Water (1.000)
7.7 5.0
330 328 330 331 396 348 336 – – 384 – 350 – 354 327 323 323 325 407
530 (0.90) 542 (0.25) 440 435 (0.03) 590 560 (0.62) 560 (0.26) 551 (0.11) 550 (0.62) 435 (0.20) 542 (0.62) 530 520 457 (0.25) (0.01) 450 441 (0.03b) 441 (0.03b) 420 (0.01b) (0.01)
[30] [27] [35] [27] [36] [27] [27] [27] [27] [27] [27] [35] [30] [27] [27] [30] [27] [27] [27] [27]
+HNRO (O)-L (HO)-LH2-AMP (HO)-L (O)-LH2 AL (HO)-L-AMP Gly–AL MeOLH2
(O)-L-AMP a b
1 mM EDTA, 0.2 M NaCl. Approximate value owing to extreme photosensitivity.
35
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39
Scheme 3. L and adenylate.
2. Fluorescence 2.1. D-LH2 and analogues The data referring to the absorption and emission of D-LH2 and analogues are presented in Tables 1 and 2. The structures of these molecules are schematically represented in Schemes 1 and 3. It has been documented that a redshift in the emission wavelength of D-LH2 occurs by increasing the temperature or lowering the pH [27–32]. A dependence on solvent polarity has also been proposed, as luciferin fluorescence at neutral pH is known to be
blue in solvents which prevent proton transfer and/or of medium polarity, such as DMSO, methanol, ice or acetone, and yellow– green in water [27]. An early study by Jung et al. [33], in which the light emitted by the phenolate form (O)-LH2 (Scheme 1) in ethanol shifted from 450 nm at 77 K to 532 nm at room temperature, backs up the correlation between temperature and fluorescence. In the same study, a similar shift was observed for the neutral (HO)-LH2 form (Scheme 1). On the other hand, Morton et al. [27] demonstrated that for the analogue 60 -methoxyLH2 (MeOLH2, Scheme 1), while the emission spectrum suggested sensitivity to solvent polarity, there was no observable variation in colour at different pH values. The authors noted, however, that the reported values were approximated due to the extreme photosensitivity of the compound, and therefore not entirely reliable. Three separate experiments [33–35] produced concordant results regarding the difference in emission wavelength between the neutral and the phenolate forms of LH2. In different mediums, the phenolate form emitted closer to red than the neutral form. This deviation seems to carry over to some luciferin analogues, namely dehydroLH2 (L, Scheme 3) and its adenylated form (LAMP, Scheme 3) [27]. In both cases, either the absorption or emis-
Table 3 Absorption and emission spectra of amino analogues of D-LH2 [37]. Solvent (ET (kcal mol1))a
NaPi Methanol (0.762) Acetonitrile (0.460) DMSO (0.444) Dimethylformamide (0.386) Dichloromethane (0.309) Chloroform (0.259) THF (0.207) Benzene (0.111) a
kabs (nm)
kem (nm)
AL
QAL
NAL
CAL
AL
QAL
NAL
CAL
349 363 362 377 363 357 357 367 356
353 363 364 380 372 361 359 372 358
322 327 333 342 329 332 329 334 327
399 414 401 415 408 393 393 403 396
523 492 478 487 479 463 466 461 437
500 479 460 468 464 438 439 438 423
465 440 425 437 425 410 412 417 400
458 461 462 472 468 461 457 462 469
Measurements at pH 7.4 in NaPi and pH 7.7 in remaining solvents.
Scheme 4. Amino analogues of D-LH2.
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J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39
Table 4 Absorption and emission spectra of OxyLH2. Solvent (ET (kcal mol1))
kabs (nm)
kem (nm) (quantum yield)
Refs.
Water (1.000) DMSO, excess PhOK Chloroform (0.259) Dichloromethane (0.309) Acetonitrile (0.460) Methanol (0.762) DMSO (0.444) Acetone (0.355)
409 475 376 369 369 372 377 367
553 570 471 465 457 457 448 436
[38] [28] [38] [38] [38] [38] [38] [38]
(0.470) (0.308) (0.231) (0.305) (0.345) (0.511) (0.277)
sion spectra of the phenolate form peaked at longer wavelengths than its protonated counterpart. In a series of studies by Erez et al. [34–36], an emission peak at approximately 590 nm in acidic aqueous solution was attributed to the zwitterion form of D-LH2 (+HNRO-), which predominated over the phenolate and neutral forms’ emission bands at 220 mM HCl [36]. Adenylation of LH2 produced a redshift in both the absorption and emission spectra in water [27]. Although L-AMP also displayed
a peak in absorption closer to red, no conclusion could be drawn in regards to its emission on account of the low quantum yield of the experiment. 2.2. Amino analogues of D-LH2 The data referring to the absorption and emission of aminoLH2 (AL) and analogues are presented in Table 3, while their structures are schematically represented in Scheme 4. A study by Nagano et al. [37] showed that the fluorescence spectrum of coumarylAL (CAL), an analogue of LH2 bearing an amino group, is practically independent of solvent polarity, whereas in general those of AL, naphtylAL (NAL) and quinolylAL (QAL) shift to red in more polar solvents, even though there is no discernible pattern when solvents of medium polarity are taken into account. CAL also differs from the other amino analogues in that it displays fluorescence at lower wavelengths than D-LH2. An emission peak at 450 nm in buffer was reported for glycineD-AL (Gly–AL) [30]. The authors proposed that the lower ionisation level of the glycine group could be responsible for the low fluores-
Table 5 Absorption and emission spectra of OxyLH2 analogues. Compound
kem (nm) (quantum yield)
Refs.
Tris–acetate buffer DMSO (0.444) Methanol (0.762) 2-Propanol (0.546) Acetonitrile (0.460) Chloroform (0.259) Benzene (0.111) p-Xylene (0.074)
485 – – – – – – –
639 630 630 624 624 588 541 541
[40] [32] [32] [32] [32] [32] [32] [32]
DMOxyLH2
Water (1.000) p-Xylene (0.074) Benzene (0.111) DMSO, excess PhOK Methanol (0.762) DMSO (0.444) 2-Propanol (0.546) Acetonitrile (0.460) Chloroform (0.259)
486 366 366 580 388 579 526 372 370
640c (0.04) 420 (0.001) 420 (0.001) 633 (0.62) 526 (0.19) 522 (0.07) 504 (0.11) 479 (0.03) 454 (0.001)
MOxyLH2 dianion
Tris–acetate buffera
440
550
DHOxyLH2
Water (1.000) DMSO (0.444) Dimethylformamide (0.386) 1-Butanol (0.586) Ethanol (0.654) Methanol (0.762) 2-Propanol (0.546) CCl4 (0.052) Hexane (0.009) Toluene (0.099) Benzene (0.111) 1,2-Dichloroethane (0.327) m-Xylene Acetone (0.355) Acetonitrile (0.460) Dichloromethane (0.309) Chloroform (0.259) Ethyl acetate (0.228)
361 367 365 363 362 361 361 367 360 366 366 359 367 358 355 348 356 359
541 451 449 441 440 440 439 439 439 439 438 436 435 434 434 434 433 432
(0.156) (0.185) (0.212) (0.42) (0.242) (0.073) (0.262) (0.676) (0.283) (0.711) (0.767) (0.610) (0.601) (0.51) (0.883) (0.40) (0.279) (0.544)
[39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39] [39]
MDMOxyLH2
p-Xylene (0.074) Water (1.000) Methanol (0.762) DMSO (0.444) 2-Propanol (0.546) Acetonitrile (0.460) Chloroform (0.259) Benzene (0.111)
368 380b 378 380 380 373 380 371
Weak 535 505 (0.12) 499 (0.008) 487 (0.02) 478 (0.02) 454 (0.002) 440 (0.001)
[32] [32] [32] [32] [32] [32] [32] [32]
( O)-DMOxyLH2
a b c
Solvent (ET (kcal mol1))
kabs (nm)
a
2 mM EDTA, 10 mM MgSO4, 1 mM dithiothreitol. Not determined accurately due to decomposition of the substrate. Fluorescence attributed to the phenolate ion.
(0.53) (0.08) (0.29) (0.26) (0.75) (0.82) (0.46)
[32] [32] [32] [28] [32] [32] [32] [32] [32] [40]
37
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39 Table 6 Bioluminescence of D-LH2, OxyLH2 and analogues. Compound
Enzyme
Bioluminescence (nm)
Refs.
D-LH2
Luc
560
[30]
AL CAL
Luc Luc Luc
596 500 578
[37] [37] [30]
Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc Luc CBRLuc CBRLuc CBRLuc CBRLuc PplGR Ppy Ppy, Luc, PplGR
615 526 564 601 556 587 570 618 615 515 560 588 559 570 450 582 499 547 548 624 560 No emission
[37] [37] [37] [37] [40] [40] [30] [40] [37] [37] [37] [37] [37] [37] [37] [37] [37] [37] [37] [41] [41] [41]
D-AL
Scheme 5. OxyLH2 analogues.
cence peak, tying the result to earlier experiments by another group on MeOLH2 and analogues. 2.3. OxyLH2 The data referring to the absorption and emission of OxyLH2 are presented in Table 4, while their structures are schematically represented in Scheme 2. As with D-LH2 and some of its analogues, the phenolate ion of OxyLH2 emits closer to red than its protonated form [32,38]. Spectroscopic studies with an ample range of solvents showed that there is no linearity between solvent polarity and emission wavelength, possibly due to the presence of several forms of OxyLH2 in solution [32,38]. In an article regarding the spectral–structural effect of the equilibria between these forms, Naumov and Kochunnoonny stated that if only the enol-enolate-keto equilibrium is taken into account, greater solvent polarity does not necessarily translate into a greater redshift in luminescence [39]. 2.4. OxyLH2 analogues The data referring to the absorption and emission of OxyLH2 analogues are presented in Table 5, while their structures are schematically represented in Scheme 5. Substitution of both hydrogen atoms in the thiazolone moiety for methyl groups shifted the emission peak towards greater wavelengths, with the phenolate form emitting closer to red than the neutral form. Further substitution of the hydroxyl in position 60 for a methoxy group caused a small blueshift [32] for 60 -methoxy-5,5-dimethylOxyLH2 (MDMOxyLH2), parallel to the effect observed for MeOLH2 [27]. This analogue showed decomposition in water, presumably due to hydrolysis of the thiazolone ring. MDMOxyLH2 and 5,5-dimethylOxyLH2 (DMOxyLH2) absorbed at around the same wavelengths in solvents of different polarity and showed considerable diversity in emission maxima. Additionally, both compounds presented very low quantum yields in less polar solvents. By comparing their emission spectra with that of bioluminescence, the authors ruled out the neutral form of OxyLH2 as the light-emitting species [32]. Ugarova proposed that while 5-methylOxyLH2 (MOxyLH2), like OxyLH2, can exist in six forms, DMOxyLH2 can only be found in its phenolate and 60 -OH forms, as the steric hindrances caused by the two methyl groups obstruct any significant changes in the adjacent C–O double bond [40]. DehydroxyOxyLH2 (DHOxyLH2) emitted at 541 nm in water and produced mostly violet light (432–451 nm) in several solvents of lower polarity. The authors attributed these results to the role of the hydroxyl anion in lowering the energy of the excited state at physiological pH, which would partially account for a redshift of the emitted light; in the absence of this group, and as a result of
DMAL DMCAL DMNAL DMQAL Enolate-OxyLH2 Enol-OxyLH2 Gly–AL Keto-OxyLH2 MAL MCAL MNAL MQAL NAL QAL MOxyLH2 dianion AL CAL NAL QAL D-DMLH2-AMP D-DMLH2
inefficient charge transfer, DHOxyLH2 can only emit orange light [39].
3. Bioluminescence 3.1. Bioluminescence emission by OxyLH2 analogues The data referring to bioluminescence emission by OxyLH2 analogues is presented in Table 6. No bioluminescence was observed for 5,5-dimethylLH2 (DMLH2). It was suggested that the addition of the two methyl groups interferes with the orientation of the carboxylate ion, ultimately preventing the adenylation of the compound due to steric hindrances. Spectroscopic studies of its adenylated form (DMLH2AMP) show that these hindrances may also have a negative influence in the oxidation step of the reaction [41]. Analysis of the Ppy- and PplGR-catalysed reactions involving D-DMLH2-AMP, of which DMOxyLH2 was the resulting compound, led the authors to conclude that red and green bioluminescence could be attributed to the keto form of this product [41]. Methylation of NAL was shown to cause a minor variation in its bioluminescence spectrum. Methyl- and dimethyl-forms of AL, QAL and CAL absorbed and emitted light at greater wavelengths [37]. 3.2. Bioluminescence emission by point mutated luciferase enzymes The data referring to bioluminescence emission by point mutated luciferase enzymes is presented in Table 7. Point mutations of residues His245, Arg215, Thr343 or Arg337 were shown to change the emission peak of Photinus pyralis luciferase towards the red region. The authors noted that there was no correlation between polarizability and emission wavelength for His245 mutants, and that the consequent spectral changes can possible be explained by a combination of effects of the protein microenvironment as a whole rather than by particular properties of residues that were introduced, pointing out that native muta-
38
J. Vieira et al. / Journal of Photochemistry and Photobiology B: Biology 117 (2012) 33–39 Table 7 Bioluminescence of wild-type and mutated luciferases [12].
For their prospective applications as substrates in bioluminescence assays, analogues of D-LH2 and OxyLH2 were analysed in regards to their spectroscopic properties under enzyme-catalysed reaction with various luciferases (most notably Luc and Ppy), and while these studies were not focused or conclusive in the search for correlations between light emission and external factors (either environmental or compound-related), they may also benefit from any significant discoveries on this subject, advancing the enhancement of the Luc system in its current medical and analytical uses. There has also been an attempt to understand the individual role of certain residues in the active site of firefly luciferase and in what capacity a modification in those positions would affect the bioluminescence reaction. Synthetic and natural mutants of luciferases effectively produced a wider range of colours than the respective wild types, and a redshifting effect was attributed to the insertion of positive residues in the active site. Nevertheless, there are no definitive answers concerning a specific position that stands out from the others, and it has been noted that the modification of residues further away from the active site is not inconsequential, which adds to the difficulty of narrowing down the search for key regions in the enzyme. Such results emphasise the current assumption that the research on the overall mechanism of colour modulation must not take the possible factors into account separately, but rather encompass all the variables that are known to influence the outcome of the light-emitting process and eventually uncover any connections between them.
Enzyme (inset: mutation)
Bioluminescence (nm)
Luciola mingrelica Ser286Lys Ser286Gln Ser286Tyr Ser286Leu His433Tyr
570 608 609 613 619 606
Luciola cruciata Ser286Asn Gly326Ser His433Tyr Pro452Ser
562 607 609 612 595
Photinus pyralis Native His245Phe His245Ala His245Arg His245Gln His245Asn His245Asp Thr343Ser Thr343Ala Arg218Lys Arg218Gln Arg218Ala Arg337Lys Arg337Gln
562 558 595 604 579 606 613 617 560 617 572 608 611 595 594
Hotaria parvula His433Tyr
568 610
Acknowledgments
Phrixothrix viviani Arg215Ser
549 589
Financial support from Fundação para a Ciência e Tecnologia (FCT, Lisbon) (Programa Operacional Temático Factores de Competitividade (COMPETE) e comparticipado pelo Fundo Comunitário Europeu (FEDER) (Project PTDC/QUI/71366/2006) is acknowledged. A Ph.D. Grant to Luís Pinto da Silva (SFRHn76612n2011), attributed by FCT, is also acknowledged.
tions of residues distant from the active site also produced redshift [12]. Mutations of Ser286 for amino acids which increased the orientation polarizability in that position were responsible for redshifts in Luciola cruciata and Luciola mingrelica luciferases [12]. Substitution of the hystidine residue in position 433 for a tyrosine caused a redshift in both enzymes [12] and produced the same result in that of Hotaria parvula [40].
4. Conclusion The effect of variations in pH and temperature on the emitted light has been confirmed in several instances and is uncontroversial in respect to D-LH2. It is known that either a decrease in pH or an increase in temperature leads to a redshift in its fluorescence wavelength. Additionally, consistent evidence pointing to a difference between the emission spectra of the neutral ligands and those of their respective phenolate ions, with the latter forms tending to emit light closer to red, further support the role of pH in colour modulation. It has been established, however, that these factors alone would not control the light-emitting process in its entirety. As other studies with analogues of D-LH2 and OxyLH2 indicated that they were affected differently by pH and temperature changes, it became clear that other aspects of the system had to be taken into account. Spectroscopic studies of LH2-related compounds in various mediums were carried out in an effort to correlate emission wavelength and solvent polarity, and despite the lack of linearity between the two, it has been proven that in general more polar solvents (i.e. water and methanol) cause a redshift compared to mediums of very low polarity.
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