ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
The Production
of Oxyluciferin
B. Department
169,616-621
J.
of Chemistry,
GATES
(19%)
During the Firefly Reaction l MARLENE
AND
Uniuersit,v
December
Light
DELUCA~
of California,
Received
Luciferase
San Diego.
La Jolla,
California
92037
16. 1974
The luciferase-product complex (E.P) was isolated from the reaction mixture after light emission had occurred. The spectral properties of the product in the E.P complex are similar to those of oxyluciferin, with a broad absorption at 385 nm. The enzyme from the complex regains full activity upon the addition of substrates. The product is not covalently bound to the enzyme and readily dissociates in the presence of 6 M urea. The isolated E. P complex was found to have 1 mol of oxyluciferin per 100,000 daltons of luciferase. No AMP could be detected in the E.P complex unless inorganic pyrophosphatase was present during the reaction. In that case 1 mol of AMP per 100,000 daltons was found. Stopped flow studies showed that an increase in 385 nm absorption occurred concomitant with light emission. Measurement of the initial rate of product formation and the rate of photon emission showed they were identical, suggesting that oxyluciferin is indeed the light-emitting product. In the initial burst of the reaction two oxyluciferin moles per 100,000 daltons of luciferase are formed. A plot of the log of the initial rate of product formation was biphasic, indicating that the first mole of product is formed at a faster rate than the second. These results are consistent with previous experiments. However, they do not resolve the question of the molecular weight of the catalytically active species.
Our tails been It
Fireflies produce light by the luciferase catalyzed oxidation of luciferin. This reaction requires ATP and Mg2+ as shown below? ,,~:)-f:~““”
+
ATP
5
present knowledge concerning the deof the organic mechanism has recently reviewed (1, 2). has been observed by several workers E LH2 AMP
+ PP!
02 9
LUCIFERIN
OXYLUCIFERIN
that during the course of bioluminescence there is an increase in absorption at 385 nm with a corresponding decrease in the absorption of luciferin at 327 nm (3, 4). Attempts to isolate the oxidized luciferin product have thus far been unsuccessful (5, 6). The product appears to be a very
’ This work was supported by Grant GB 36014 from the National Science Foundation. 2Person to whom correspondence should be sent. 3 Abbreviations used are: LH,, luciferin; E, luciferase; LH,AMP, luciferyl adenylate; E-P, luciferaseoxyluciferin (product) complex; E. LAMP, leuciferasedehydroluciferin-AMP complex. 616 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
OXYLUCIFERIN
PRODUCTION
reactive molecule because it is rapidly converted to several other compounds (6). Recently, Goto and coworkers (7) have synthesized 2-(6’-hydroxybenzothiazol-2’yl)-4-hydroxythiazole, “oxyluciferin.” This compound shows a maximum absorbance at 382 nm at pH 7.0 and its fluorescence spectrum in dimethyl sulfoxide is the same as the luciferase bioluminescence emission spectrum. They proposed that oxyluciferin is the emittor in both the bioluminescent and chemiluminescent oxidations. They also were able to isolate a more stable derivative of this compound, having similar spectral properties, from firefly lanterns and spent chemiluminescent reaction mixtures (8). This report extends these studies by following the formation of oxyluciferin using purified luciferase to catalyze the reaction. We have determined the rate of production of oxyluciferin and compared this to the rate of light production. We also measured the stoichiometry of the amount of product formed during the light flash to the amount of enzyme present by following the changes in absorption at 385 nm. Our results support Goto’s conclusion that oxyluciferin is indeed the product of the bioluminescent reaction. MATERIALS
AND
METHODS
Firefly luciferase was prepared as described by Green and McElroy (1956) (9). Luciferin was synthesized by Dr. Lemuel Bowie according to the method of Seto et al. (1963) (10). Synthetic LH,-AMP was prepared as described by Morton et al. (1969) (11). [“Cl ATP (30.9 pCi/pm) was obtained from New England Nuclear Corporation. Inorganic pyrophosphatase (Type III) was a product of Sigma. The stopped-flow experiments were performed with an Aminco-Morrow Stopped-Flow Spectrophotometer. The instrument was equipped with an IP28 phototube, a xenon light source. and a Tetronix Model RM564 storage oscilloscope equipped with a type 2A63 differential amplifier and a type 2B67 time base, as well as with a signal integrator for following total light production. The reactions were initiated by mixing luciferin, ATP, and MgSO, held in the one syringe with luciferase contained in the other syringe. Both syringes contained 0.1 M Tris-HCI (pH 7.8). The final concentrations of the reactants were: 0.2 mM luciferin; 2 mM ATP; 5 mM MgSO,; and lo-14 pM luciferase. The mixing time of the stopped-flow cell was less than 2 ms. All stopped-flow experiments were performed at 25°C unless otherwise stated.
617
BY LUCIFERASE
For isolation of the enzyme-product complex, the experimental conditions were the following: The reaction mixture contained 2.5 mM ATP. 5 mM MaSO,, 1.25 rnM luciferin. 622133 pM luciferase and 0.1 M Tris-HCl (pH 7.8) in a total volume of 0.4 ml. After 15 min of incubation at room temperature, the spent reaction was extensively dialyzed vs 0.1 M potassium phosphate or 0.1 M Trls-HCI (pH 7.8) at 4°C until the dialyzate was no longer fluorescent, 24 h. When LH,-AMP was used instead of ATP, M&O,, and luciferin, luciferase was titrated with the intermediate until further additions no longer produced light. A 2 x 52 cm column of Sephadex G-100 fine was used for the molecular weight determination of E.P. The column was calibrated by measuring the elution volume of aldolase (158.000 daltons), bovine serum albumin (68,000 daltons). and chymotrypsin A (25,000 daltoms). The column was equilibrated and eluted with 0.1 M potassium phosphate (pH 7.0). RESULTS
AND
DISCUSSION
Enzyme-Product Complex Luciferase was reacted with lo- to %Ofold excess of luciferin, ATP and MgSO, for about 15 min. Excess substrates were removed either by exhaustive dialysis or by passage through a Sephadex G-25 column. The absorption spectrum of the isolated E-P was found to have a broad maximum at 385 nm (Fig. 1). The uncorrected excitation and fluorescence spectra of E-P are shown in Fig. 2. These spectra are consistent with the spectral maximum reported by Goto and coworkers for oxyluciferin (7). However, the fluorescence emission of the product on the enzyme (Xmax 523 nm) does not agree with the firefly (Photinus pyralis) bioluminescence spectra (A,,,,, 562 nm) (1). This could be explained bv considering the product to be in a different environment on the enzyme after the bioluminescent reaction than during it. Such a difference could be attributed to a conformational change of luciferase (see later discussion). Assuming the molar extinction coefficient of the product at 385 nm to be 11,500 (7), the ratio of product to enzyme is in the range of 1.1 F 0.2 mol of product per 100,000 daltons. A similar experiment was performed by incubating luciferase with an excess of synthetic LH,-AMP and the ratio of product to enzyme again was found to be 0.9/100,000 daltons. McElroy has noted that a blue pigment appeared when a spent reaction was allowed to stand at room temperature for 64
618
GATES
I 250
I 280
AND
DELUCA
1 340
310
I 370
WAVELENGTH
FIG. 1. Absorption phosphate (pH 7.8);
(.
400
1 430
spectra of native luciferase (---) and isolated . .) E.P after dialysis against 6 M urea is (-).
300
FIG. 2. Excitation
380
460 380 WAVELENGTH
and emission
spectra
h following the bioluminescent flash. This was attributed to the product slowly becoming covalently attached to the enzyme (9). We reacted luciferase with excess substrates as above, and allowed the spent reaction to stand at room temperature for 24 h until the blue pigment appeared. After the reaction mix was dialyzed against 6 M urea, there was no indication of any bound product (Fig. 1). If the spent reaction mixture was dialyzed against 6 M urea immediately after the light flash rather than buffer, no product remained with the enzyme as judged by the absence of any absorption at 385 nm (Fig. 1). These experiments demonstrate that the product is not covalently bound to luciferase. This agrees with the observation made by Goto that oxyluciferin is a competitive inhibitor of the light reaction (12). The molecular weight of E*P from the
460
( nm)
460 --
E .P in 0.1
540 -A
M
potassium
620
(nmi
of E-P
in 0.1 M Tris-HCl
(pH
7.8)
spent reaction was determined by chromatography on a calibrated Sephadex G-100 column. It was found to be approximately 47,000 daltons, the same as that found for the native enzyme under these conditions. Since it is thought that AMP is bound to the E.P during light emission (13), it was of interest to know if the isolated E. P contained any bound AMP. To determine this, luciferase was incubated with 20-fold excess luciferin, [14C]ATP and MgCl, and E.P complex was separated from the other reagents on a Sephadex G-25 column. The results of this experiment can be seen in Fig. 3b. Although the isolated enzyme was found to have 1.1 moles of oxyluciferin per 100,000 daltons, essentially no radioactivity was associated with the E.P. Assuming that one mole of [l*C]AMP remained with the enzyme, there should have been 1720 cpm. The observed counts are less than 6%
OXYLUCIFERIN
PRODUCTION
of this amount. However if the same experiment is done in the presence of inorganic pyrophosphatase, it is found that the E*P complex contains 0.7 mol of AMP per 100,000 daltons (Fig. 3a). Thus, it appears that removing inorganic pyrophosphate from the medium allows the binding of AMP to the E.P. The significance of these observations is not clear at the present time. When the isolated E.P complex was assayed for light emission by adding luciferin, Mg2+, and injecting ATP, we found that the complex becomes fully active based on the flash height. When E.P is formed in the light reaction by mixing excess substrates with luciferase, the enzyme is rapidly inhibited from producing light (Fig. 4). However, one can isolate an (,
1,)
n
I,,
,/18
a 14 1
BY LUCIFERASE
E*P complex, containing one mole of oxyluciferin per 100,000 daltons which has regained full activity with respect to light emission when it is mixed with fresh substrates. If oxyluciferin is a true competitive inhibitor, the above results make it difficult to understand why the enzyme is so rapidly inhibited by small amounts of product in the presence of a large excess of luciferin. A possible explanation could be that the E.P formed in the light reaction is different from the isolated E*P complex. That the difference could be due to a conformational change has been suggested by fluorescence spectra of E-P (see above). DeLuca has demonstrated by tritium-hydrogen exchange and optical rotary dispersion studies that a large conformational change in luciferase occurred in the presence of substrates (14). In addition, thermodynamic calculations indicate that conformational changes may occur during the lag prior to light emission (15). Thus, it is logical to assume that conformational changes would have to occur in order to regenerate the native form that could again bind substrates. The presence of products in the spent reaction might make this more difficult. In this respect it is easier to understand the low turnover rate of the enzyme after the flash. Stoichiometry
106
FIG. 3. Sephadex G-25 chromatography of 0.4 ml samples of the Iuciferase reaction 5 min after the light flash. A column of Sephadex G-75 fine (1 x 25 cm) was equilibrated with 0.01 M Tris-HCl (pH 7.5). The flow rate was 27 ml per h and 1.0.ml fractions were collected (a) The reaction mixture contained 5 mM 0.75 mM LH,, 70 FM luciferase, 0.1 M M&O,, Tris-HCl (pH 7.5), 2.5 mM [“C]ATP (0.1 &i/mM), and 1.7 units of inorganic pyrophosphatase (bl The same as (a) except no inorganic pyrophosphatase present.
619
and Kinetics Formation
of Oxyluciferin
If oxyluciferin is the immediate product of the light reaction, it must be formed at a rate equal to that of the emission of light. The initial rate of light production and the rate of formation of oxyluciferin were measured in a stopped-flow apparatus. To detect the light emission, the light source of the instrument was turned off, and the bioluminescence was followed after rapid mixing of the enzyme with the substrates. The result of a typical experiment is shown in Fig. 4a. The flash height shows the amount of light emitted as a function of time, whereas the lower dashed curve represents the total integrated light. The flash height peaks at 0.3 second after initiation of the reaction (15), and decays exponentially to a low level of emission
620
GATES PIE
LIGHT
INTENSITY
AND IV1
2 3 m 2
TOTAL
LIGHT
IVI
FK. 4. Stopped-flow oscilloscope tracings of the luciferase reaction with excess substrates. (a) L@t emission. Solid line indicates light intensity, whereas the dashed line represents the total emitted light obtained by integration. (b) Oryluciferin formation. P/E represents the number of moles of oxyluciferin produced per 100,000 daltons of enzyme, calculated from the increase in absorbance at 385 nm.
within 5 s. This rapid decay of the bioluminescence is thought to be due to the inhibition of luciferase by oxyluciferin (16). To measure the appearance of product, the light source was turned on and the monochrometer was set at 385 nm. A Klett No. 42 filter was placed in front of the phototube to exclude any bioluminescent light. Fig. 4b shows a graph of the rate of formation of oxyluciferin, as indicated by the increase in absorption at 385 nm, under enzyme-limiting conditions. When the very early phases of the reaction are monitored, one observes a lag of 25 ms before any increase in absorption at 385 nm is detected. A similar lag is observed in the emission of light (15). From the amplitude of the burst, we calculated that two moles of product are formed per mole of enzyme (100,000 molecular weight). This number was arrived at by extrapolation from the steady state portion of the curve at 5 s (Fig. 1) back to the ordinate (17). A plot of the log of the percentage of unreacted enzyme vs. time (Fig. 5) was biphasic, indicating that the first mole of product is formed faster than the second. Although there are two binding sites for LH, per 100,000 daltons (18), apparently only one of these is operative in the initial fast reaction. This indicates that the lucif-
DELUCA
erin binding sites are non-equivalent in terms of activity. To compare the rate of light production (Fig. 4a) and the rate of product formation (Fig. 4b), the data were expressed as first order reactions (Fig. 5). This analysis revealed the rates to be the same. These experiments demonstrate that oxyluciferin is formed at the same rate as photon emission occurs and support the suggestion that it is the product of the light reaction. It is currently thought that the molecular weight of the active species of luciferase is 50,000 daltons. Though at high concentrations luciferase does aggregate in low ionic strength solvents (19). Based on flash height measurements, Denburg showed that the enzyme exhibited no change in
‘0°1
IO
I
2 TIME
3 (seconds)
4
5
6
FIG. 5. Log plot of pre-steady-state liberation of oxyluciferin 0 -0 and integrated light O-O vs time in the luciferase catalyzed reaction. The data were obtained from stopped-flow oscilloscope tracings of oxyluciferin production and integrated light following mixing of luciferase with substrates at 12°C. The data are expressed as the log of the percentage of unreacted enzyme VS. time. The amplitude of the oscilloscope tracing at the time when 2 mol of oxyluciferin .were formed per 100,000 daltons luciferase was taken as 100% reacted.
OXYLUCIFERIN
PRODUCTION
specific activity as the molecular weight of enzyme increased from 50,000-110,000 (19). In addition, in the same paper he reported the molecular weight of the E. LAMP’ to be 50,000 by Sephadex chromatography. Also, we found that the molecular weight of the isolated E . P is 50,000 daltons by Sephadex G-100 chromatography. However, some data in this report indicates that the molecular weight of the active species could be 100,000 daltons: (1) there is a rapid formation of one product/lOO,OOO daltons in the burst; and (2) the isolated E. P complex shows only 1 oxyluciferin/lOO,OOO daltons. Previous data which indicates that the active luciferase might be a dimer of two 50,000 dalton subunits includes: the binding of only 1 mol of Mg”-ATP/lOO,OOO daltons5 (20), and the ability to form only one E LAMP at a time (19). If the molecular weight of the active species is 100,000 daltons, this would include luciferase in the group of enzymes known to exhibit half site reactivity such as horse-liver alcohol dehydrogenase (20), Escherichia co/i alkaline phosphatase (21) and yeast glyceraldehyde 3-phosphate dehydrogenase (22). Further research to resolve this problem is in progress. ACKNOWLEDGMENTS The author would like to thank Doctors Johannes Everse and B. J. Conner for many helpful discussions on this work and Dr. N. 0. Kaplan for use of his stopped-flow apparatus.
BY LUCIFERASE
6.
7. 8. 9. 10. 11. 12.
13.
14. 15. 16.
17.
REFERENCES 1. MCELROY, W. D., AND DELUCA, M. (1974) in Chemiluminescence and Bioluminescence (Cormier, M. J., Hercules, D. M., and Lee, J., eds.), pp. 285-311, Plenum, New York. 2. DELucA, M., AND DEMPSEY, M. (1974) in Chemiluminescence and Bioluminescence (Cormier, M. J., Hercules, D. M., and Lee, J., eds.). pp. 345-355, Plenum, New York.
18. 19. 20. 21. 22.
4 Dehydroluciferin is an analog of luciferin a competitive inhibitor of luciferase. 5Mg 2+ ATP is the active form of the
(20).
which substrate
is 23.
621
MCELKOY, W. D., AND GREEN, A. (1956) Arch. Biochem. Biophys. 64, 257-271. HOPKINS, T. A. (1968). Ph.D. Thesis, Johns Hopkins University. SELIGER, H. H., AND MCELROY. W. D. (19661 in Bioluminescence in Progress (Johnson, F. H., and Haneda, Y., eds.1. pp. 429-458, Princeton University Press, Princeton, New Jersey. PLANT, P. J., WHITE, E. H., AND MCELROY, W. D. (1968) Biochem. Biophgs. Res. Comm. 31, 9% 103. SVZUKI, N., SATO, M., NISHIKA~A, K., AND GOTO, T. (1969) Tetr. Lett. 53, 4683-4684. SUZVKI, N., AND GOTO, T. (1971) Tetr. Lett. 22, 2021-2022. GREEN, A., AND MCELROY, W. D. (1956) Biochim Biophys. Acta. 20, 170-176. SETO, S., OZURA, K., AND NISHIJAMA, Y. (1963) Bull. Chem. Sot. Jap. 36, 331-333. MORTON, R. A., HOPKINS, T. A., AND SELIGER, H. H. (1969) Biochemistry 8, 1598-1607. GOTO, T., KUBBOTA, I., SUZUKI, N., AND KISHI, Y. (19741 in Chemiluminescence and Bioluminescence (Cormier, M. J., Hercules, D. M.. and Lee, J., eds.), pp. 325-335, Plenum, New York. DELUCA, M., LEONARD, N. J., GATES, B. J., AND MCELROY, W. D. (1973) Proc. Nat. Acad. Sci. USA 70, 1664-1666. DELUCA, M., AP*‘D MARSH, M. (1967) Arch. Biothem, Biophys. 121, 233-240. DELUCA, M., AND MCELROY, W. D. (1974) Biochemistry 13, 921-925. MCELROY, W. D., AND SELIGER, H. H. (1961) in Light and Life (McElroy, W. D. and Glass, B., eds.1, pp. 219-254, Johns Hopkins Press, Baltimore. BENDER, M. L., KEZD~, F. J., AND WEDLER, F. C. (1967) J. Chem. Educ. 44,84-88. DENBCRG, J. L., LEE, R. T., AND MCELROY, W. D. (1969) Arch. Biochem. Biophys. 134, 381-394. DENBURG. J. L., AND MCELROY, W. D. (1970) Biochemistry 9, 4619-4624. LEE, R. T., DENBURG, J. L. AND MCELROY, W. D. (1970) Arch. Biochem. Biophys. 141, 38-52. LUISI, P. L., AND BIGNEWL, E. (19741 J. Mol. Biol. 88, 00-018. CHAPPELET-TORDO, D., IWASTSUBO, M., AND LAZDUNSKL M. (1974) Biochemistry 13, 37543762. STALLCUP, W. B., AND KOSHLAND, D. E., JR. (1973) J. Mol. Biol. 80, 41-62.
OF BIOCHEMISTRY
AND BIOPHYSICS
The Production
of Oxyluciferin
B. Department
169,616-621
J.
of Chemistry,
GATES
(19%)
During the Firefly Reaction l MARLENE
AND
Uniuersit,v
December
Light
DELUCA~
of California,
Received
Luciferase
San Diego.
La Jolla,
California
92037
16. 1974
The luciferase-product complex (E.P) was isolated from the reaction mixture after light emission had occurred. The spectral properties of the product in the E.P complex are similar to those of oxyluciferin, with a broad absorption at 385 nm. The enzyme from the complex regains full activity upon the addition of substrates. The product is not covalently bound to the enzyme and readily dissociates in the presence of 6 M urea. The isolated E. P complex was found to have 1 mol of oxyluciferin per 100,000 daltons of luciferase. No AMP could be detected in the E.P complex unless inorganic pyrophosphatase was present during the reaction. In that case 1 mol of AMP per 100,000 daltons was found. Stopped flow studies showed that an increase in 385 nm absorption occurred concomitant with light emission. Measurement of the initial rate of product formation and the rate of photon emission showed they were identical, suggesting that oxyluciferin is indeed the light-emitting product. In the initial burst of the reaction two oxyluciferin moles per 100,000 daltons of luciferase are formed. A plot of the log of the initial rate of product formation was biphasic, indicating that the first mole of product is formed at a faster rate than the second. These results are consistent with previous experiments. However, they do not resolve the question of the molecular weight of the catalytically active species.
Our tails been It
Fireflies produce light by the luciferase catalyzed oxidation of luciferin. This reaction requires ATP and Mg2+ as shown below? ,,~:)-f:~““”
+
ATP
5
present knowledge concerning the deof the organic mechanism has recently reviewed (1, 2). has been observed by several workers E LH2 AMP
+ PP!
02 9
LUCIFERIN
OXYLUCIFERIN
that during the course of bioluminescence there is an increase in absorption at 385 nm with a corresponding decrease in the absorption of luciferin at 327 nm (3, 4). Attempts to isolate the oxidized luciferin product have thus far been unsuccessful (5, 6). The product appears to be a very
’ This work was supported by Grant GB 36014 from the National Science Foundation. 2Person to whom correspondence should be sent. 3 Abbreviations used are: LH,, luciferin; E, luciferase; LH,AMP, luciferyl adenylate; E-P, luciferaseoxyluciferin (product) complex; E. LAMP, leuciferasedehydroluciferin-AMP complex. 616 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
OXYLUCIFERIN
PRODUCTION
reactive molecule because it is rapidly converted to several other compounds (6). Recently, Goto and coworkers (7) have synthesized 2-(6’-hydroxybenzothiazol-2’yl)-4-hydroxythiazole, “oxyluciferin.” This compound shows a maximum absorbance at 382 nm at pH 7.0 and its fluorescence spectrum in dimethyl sulfoxide is the same as the luciferase bioluminescence emission spectrum. They proposed that oxyluciferin is the emittor in both the bioluminescent and chemiluminescent oxidations. They also were able to isolate a more stable derivative of this compound, having similar spectral properties, from firefly lanterns and spent chemiluminescent reaction mixtures (8). This report extends these studies by following the formation of oxyluciferin using purified luciferase to catalyze the reaction. We have determined the rate of production of oxyluciferin and compared this to the rate of light production. We also measured the stoichiometry of the amount of product formed during the light flash to the amount of enzyme present by following the changes in absorption at 385 nm. Our results support Goto’s conclusion that oxyluciferin is indeed the product of the bioluminescent reaction. MATERIALS
AND
METHODS
Firefly luciferase was prepared as described by Green and McElroy (1956) (9). Luciferin was synthesized by Dr. Lemuel Bowie according to the method of Seto et al. (1963) (10). Synthetic LH,-AMP was prepared as described by Morton et al. (1969) (11). [“Cl ATP (30.9 pCi/pm) was obtained from New England Nuclear Corporation. Inorganic pyrophosphatase (Type III) was a product of Sigma. The stopped-flow experiments were performed with an Aminco-Morrow Stopped-Flow Spectrophotometer. The instrument was equipped with an IP28 phototube, a xenon light source. and a Tetronix Model RM564 storage oscilloscope equipped with a type 2A63 differential amplifier and a type 2B67 time base, as well as with a signal integrator for following total light production. The reactions were initiated by mixing luciferin, ATP, and MgSO, held in the one syringe with luciferase contained in the other syringe. Both syringes contained 0.1 M Tris-HCI (pH 7.8). The final concentrations of the reactants were: 0.2 mM luciferin; 2 mM ATP; 5 mM MgSO,; and lo-14 pM luciferase. The mixing time of the stopped-flow cell was less than 2 ms. All stopped-flow experiments were performed at 25°C unless otherwise stated.
617
BY LUCIFERASE
For isolation of the enzyme-product complex, the experimental conditions were the following: The reaction mixture contained 2.5 mM ATP. 5 mM MaSO,, 1.25 rnM luciferin. 622133 pM luciferase and 0.1 M Tris-HCl (pH 7.8) in a total volume of 0.4 ml. After 15 min of incubation at room temperature, the spent reaction was extensively dialyzed vs 0.1 M potassium phosphate or 0.1 M Trls-HCI (pH 7.8) at 4°C until the dialyzate was no longer fluorescent, 24 h. When LH,-AMP was used instead of ATP, M&O,, and luciferin, luciferase was titrated with the intermediate until further additions no longer produced light. A 2 x 52 cm column of Sephadex G-100 fine was used for the molecular weight determination of E.P. The column was calibrated by measuring the elution volume of aldolase (158.000 daltons), bovine serum albumin (68,000 daltons). and chymotrypsin A (25,000 daltoms). The column was equilibrated and eluted with 0.1 M potassium phosphate (pH 7.0). RESULTS
AND
DISCUSSION
Enzyme-Product Complex Luciferase was reacted with lo- to %Ofold excess of luciferin, ATP and MgSO, for about 15 min. Excess substrates were removed either by exhaustive dialysis or by passage through a Sephadex G-25 column. The absorption spectrum of the isolated E-P was found to have a broad maximum at 385 nm (Fig. 1). The uncorrected excitation and fluorescence spectra of E-P are shown in Fig. 2. These spectra are consistent with the spectral maximum reported by Goto and coworkers for oxyluciferin (7). However, the fluorescence emission of the product on the enzyme (Xmax 523 nm) does not agree with the firefly (Photinus pyralis) bioluminescence spectra (A,,,,, 562 nm) (1). This could be explained bv considering the product to be in a different environment on the enzyme after the bioluminescent reaction than during it. Such a difference could be attributed to a conformational change of luciferase (see later discussion). Assuming the molar extinction coefficient of the product at 385 nm to be 11,500 (7), the ratio of product to enzyme is in the range of 1.1 F 0.2 mol of product per 100,000 daltons. A similar experiment was performed by incubating luciferase with an excess of synthetic LH,-AMP and the ratio of product to enzyme again was found to be 0.9/100,000 daltons. McElroy has noted that a blue pigment appeared when a spent reaction was allowed to stand at room temperature for 64
618
GATES
I 250
I 280
AND
DELUCA
1 340
310
I 370
WAVELENGTH
FIG. 1. Absorption phosphate (pH 7.8);
(.
400
1 430
spectra of native luciferase (---) and isolated . .) E.P after dialysis against 6 M urea is (-).
300
FIG. 2. Excitation
380
460 380 WAVELENGTH
and emission
spectra
h following the bioluminescent flash. This was attributed to the product slowly becoming covalently attached to the enzyme (9). We reacted luciferase with excess substrates as above, and allowed the spent reaction to stand at room temperature for 24 h until the blue pigment appeared. After the reaction mix was dialyzed against 6 M urea, there was no indication of any bound product (Fig. 1). If the spent reaction mixture was dialyzed against 6 M urea immediately after the light flash rather than buffer, no product remained with the enzyme as judged by the absence of any absorption at 385 nm (Fig. 1). These experiments demonstrate that the product is not covalently bound to luciferase. This agrees with the observation made by Goto that oxyluciferin is a competitive inhibitor of the light reaction (12). The molecular weight of E*P from the
460
( nm)
460 --
E .P in 0.1
540 -A
M
potassium
620
(nmi
of E-P
in 0.1 M Tris-HCl
(pH
7.8)
spent reaction was determined by chromatography on a calibrated Sephadex G-100 column. It was found to be approximately 47,000 daltons, the same as that found for the native enzyme under these conditions. Since it is thought that AMP is bound to the E.P during light emission (13), it was of interest to know if the isolated E. P contained any bound AMP. To determine this, luciferase was incubated with 20-fold excess luciferin, [14C]ATP and MgCl, and E.P complex was separated from the other reagents on a Sephadex G-25 column. The results of this experiment can be seen in Fig. 3b. Although the isolated enzyme was found to have 1.1 moles of oxyluciferin per 100,000 daltons, essentially no radioactivity was associated with the E.P. Assuming that one mole of [l*C]AMP remained with the enzyme, there should have been 1720 cpm. The observed counts are less than 6%
OXYLUCIFERIN
PRODUCTION
of this amount. However if the same experiment is done in the presence of inorganic pyrophosphatase, it is found that the E*P complex contains 0.7 mol of AMP per 100,000 daltons (Fig. 3a). Thus, it appears that removing inorganic pyrophosphate from the medium allows the binding of AMP to the E.P. The significance of these observations is not clear at the present time. When the isolated E.P complex was assayed for light emission by adding luciferin, Mg2+, and injecting ATP, we found that the complex becomes fully active based on the flash height. When E.P is formed in the light reaction by mixing excess substrates with luciferase, the enzyme is rapidly inhibited from producing light (Fig. 4). However, one can isolate an (,
1,)
n
I,,
,/18
a 14 1
BY LUCIFERASE
E*P complex, containing one mole of oxyluciferin per 100,000 daltons which has regained full activity with respect to light emission when it is mixed with fresh substrates. If oxyluciferin is a true competitive inhibitor, the above results make it difficult to understand why the enzyme is so rapidly inhibited by small amounts of product in the presence of a large excess of luciferin. A possible explanation could be that the E.P formed in the light reaction is different from the isolated E*P complex. That the difference could be due to a conformational change has been suggested by fluorescence spectra of E-P (see above). DeLuca has demonstrated by tritium-hydrogen exchange and optical rotary dispersion studies that a large conformational change in luciferase occurred in the presence of substrates (14). In addition, thermodynamic calculations indicate that conformational changes may occur during the lag prior to light emission (15). Thus, it is logical to assume that conformational changes would have to occur in order to regenerate the native form that could again bind substrates. The presence of products in the spent reaction might make this more difficult. In this respect it is easier to understand the low turnover rate of the enzyme after the flash. Stoichiometry
106
FIG. 3. Sephadex G-25 chromatography of 0.4 ml samples of the Iuciferase reaction 5 min after the light flash. A column of Sephadex G-75 fine (1 x 25 cm) was equilibrated with 0.01 M Tris-HCl (pH 7.5). The flow rate was 27 ml per h and 1.0.ml fractions were collected (a) The reaction mixture contained 5 mM 0.75 mM LH,, 70 FM luciferase, 0.1 M M&O,, Tris-HCl (pH 7.5), 2.5 mM [“C]ATP (0.1 &i/mM), and 1.7 units of inorganic pyrophosphatase (bl The same as (a) except no inorganic pyrophosphatase present.
619
and Kinetics Formation
of Oxyluciferin
If oxyluciferin is the immediate product of the light reaction, it must be formed at a rate equal to that of the emission of light. The initial rate of light production and the rate of formation of oxyluciferin were measured in a stopped-flow apparatus. To detect the light emission, the light source of the instrument was turned off, and the bioluminescence was followed after rapid mixing of the enzyme with the substrates. The result of a typical experiment is shown in Fig. 4a. The flash height shows the amount of light emitted as a function of time, whereas the lower dashed curve represents the total integrated light. The flash height peaks at 0.3 second after initiation of the reaction (15), and decays exponentially to a low level of emission
620
GATES PIE
LIGHT
INTENSITY
AND IV1
2 3 m 2
TOTAL
LIGHT
IVI
FK. 4. Stopped-flow oscilloscope tracings of the luciferase reaction with excess substrates. (a) L@t emission. Solid line indicates light intensity, whereas the dashed line represents the total emitted light obtained by integration. (b) Oryluciferin formation. P/E represents the number of moles of oxyluciferin produced per 100,000 daltons of enzyme, calculated from the increase in absorbance at 385 nm.
within 5 s. This rapid decay of the bioluminescence is thought to be due to the inhibition of luciferase by oxyluciferin (16). To measure the appearance of product, the light source was turned on and the monochrometer was set at 385 nm. A Klett No. 42 filter was placed in front of the phototube to exclude any bioluminescent light. Fig. 4b shows a graph of the rate of formation of oxyluciferin, as indicated by the increase in absorption at 385 nm, under enzyme-limiting conditions. When the very early phases of the reaction are monitored, one observes a lag of 25 ms before any increase in absorption at 385 nm is detected. A similar lag is observed in the emission of light (15). From the amplitude of the burst, we calculated that two moles of product are formed per mole of enzyme (100,000 molecular weight). This number was arrived at by extrapolation from the steady state portion of the curve at 5 s (Fig. 1) back to the ordinate (17). A plot of the log of the percentage of unreacted enzyme vs. time (Fig. 5) was biphasic, indicating that the first mole of product is formed faster than the second. Although there are two binding sites for LH, per 100,000 daltons (18), apparently only one of these is operative in the initial fast reaction. This indicates that the lucif-
DELUCA
erin binding sites are non-equivalent in terms of activity. To compare the rate of light production (Fig. 4a) and the rate of product formation (Fig. 4b), the data were expressed as first order reactions (Fig. 5). This analysis revealed the rates to be the same. These experiments demonstrate that oxyluciferin is formed at the same rate as photon emission occurs and support the suggestion that it is the product of the light reaction. It is currently thought that the molecular weight of the active species of luciferase is 50,000 daltons. Though at high concentrations luciferase does aggregate in low ionic strength solvents (19). Based on flash height measurements, Denburg showed that the enzyme exhibited no change in
‘0°1
IO
I
2 TIME
3 (seconds)
4
5
6
FIG. 5. Log plot of pre-steady-state liberation of oxyluciferin 0 -0 and integrated light O-O vs time in the luciferase catalyzed reaction. The data were obtained from stopped-flow oscilloscope tracings of oxyluciferin production and integrated light following mixing of luciferase with substrates at 12°C. The data are expressed as the log of the percentage of unreacted enzyme VS. time. The amplitude of the oscilloscope tracing at the time when 2 mol of oxyluciferin .were formed per 100,000 daltons luciferase was taken as 100% reacted.
OXYLUCIFERIN
PRODUCTION
specific activity as the molecular weight of enzyme increased from 50,000-110,000 (19). In addition, in the same paper he reported the molecular weight of the E. LAMP’ to be 50,000 by Sephadex chromatography. Also, we found that the molecular weight of the isolated E . P is 50,000 daltons by Sephadex G-100 chromatography. However, some data in this report indicates that the molecular weight of the active species could be 100,000 daltons: (1) there is a rapid formation of one product/lOO,OOO daltons in the burst; and (2) the isolated E. P complex shows only 1 oxyluciferin/lOO,OOO daltons. Previous data which indicates that the active luciferase might be a dimer of two 50,000 dalton subunits includes: the binding of only 1 mol of Mg”-ATP/lOO,OOO daltons5 (20), and the ability to form only one E LAMP at a time (19). If the molecular weight of the active species is 100,000 daltons, this would include luciferase in the group of enzymes known to exhibit half site reactivity such as horse-liver alcohol dehydrogenase (20), Escherichia co/i alkaline phosphatase (21) and yeast glyceraldehyde 3-phosphate dehydrogenase (22). Further research to resolve this problem is in progress. ACKNOWLEDGMENTS The author would like to thank Doctors Johannes Everse and B. J. Conner for many helpful discussions on this work and Dr. N. 0. Kaplan for use of his stopped-flow apparatus.
BY LUCIFERASE
6.
7. 8. 9. 10. 11. 12.
13.
14. 15. 16.
17.
REFERENCES 1. MCELROY, W. D., AND DELUCA, M. (1974) in Chemiluminescence and Bioluminescence (Cormier, M. J., Hercules, D. M., and Lee, J., eds.), pp. 285-311, Plenum, New York. 2. DELucA, M., AND DEMPSEY, M. (1974) in Chemiluminescence and Bioluminescence (Cormier, M. J., Hercules, D. M., and Lee, J., eds.). pp. 345-355, Plenum, New York.
18. 19. 20. 21. 22.
4 Dehydroluciferin is an analog of luciferin a competitive inhibitor of luciferase. 5Mg 2+ ATP is the active form of the
(20).
which substrate
is 23.
621
MCELKOY, W. D., AND GREEN, A. (1956) Arch. Biochem. Biophys. 64, 257-271. HOPKINS, T. A. (1968). Ph.D. Thesis, Johns Hopkins University. SELIGER, H. H., AND MCELROY. W. D. (19661 in Bioluminescence in Progress (Johnson, F. H., and Haneda, Y., eds.1. pp. 429-458, Princeton University Press, Princeton, New Jersey. PLANT, P. J., WHITE, E. H., AND MCELROY, W. D. (1968) Biochem. Biophgs. Res. Comm. 31, 9% 103. SVZUKI, N., SATO, M., NISHIKA~A, K., AND GOTO, T. (1969) Tetr. Lett. 53, 4683-4684. SUZVKI, N., AND GOTO, T. (1971) Tetr. Lett. 22, 2021-2022. GREEN, A., AND MCELROY, W. D. (1956) Biochim Biophys. Acta. 20, 170-176. SETO, S., OZURA, K., AND NISHIJAMA, Y. (1963) Bull. Chem. Sot. Jap. 36, 331-333. MORTON, R. A., HOPKINS, T. A., AND SELIGER, H. H. (1969) Biochemistry 8, 1598-1607. GOTO, T., KUBBOTA, I., SUZUKI, N., AND KISHI, Y. (19741 in Chemiluminescence and Bioluminescence (Cormier, M. J., Hercules, D. M.. and Lee, J., eds.), pp. 325-335, Plenum, New York. DELUCA, M., LEONARD, N. J., GATES, B. J., AND MCELROY, W. D. (1973) Proc. Nat. Acad. Sci. USA 70, 1664-1666. DELUCA, M., AP*‘D MARSH, M. (1967) Arch. Biothem, Biophys. 121, 233-240. DELUCA, M., AND MCELROY, W. D. (1974) Biochemistry 13, 921-925. MCELROY, W. D., AND SELIGER, H. H. (1961) in Light and Life (McElroy, W. D. and Glass, B., eds.1, pp. 219-254, Johns Hopkins Press, Baltimore. BENDER, M. L., KEZD~, F. J., AND WEDLER, F. C. (1967) J. Chem. Educ. 44,84-88. DENBCRG, J. L., LEE, R. T., AND MCELROY, W. D. (1969) Arch. Biochem. Biophys. 134, 381-394. DENBURG. J. L., AND MCELROY, W. D. (1970) Biochemistry 9, 4619-4624. LEE, R. T., DENBURG, J. L. AND MCELROY, W. D. (1970) Arch. Biochem. Biophys. 141, 38-52. LUISI, P. L., AND BIGNEWL, E. (19741 J. Mol. Biol. 88, 00-018. CHAPPELET-TORDO, D., IWASTSUBO, M., AND LAZDUNSKL M. (1974) Biochemistry 13, 37543762. STALLCUP, W. B., AND KOSHLAND, D. E., JR. (1973) J. Mol. Biol. 80, 41-62.
