Studies on the bioluminescence of renilla reniformis V. Absorption and fluorescence characteristics of chromatographically pure luciferin

386

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 45 192

STUDIES ON T H E B I O L U M I N E S C E N C E OF RENILLA RENIFORMIS V. A B S O R P T I O N AND FLUORESCENCE CHARACTERISTICS OF CHROMATOGRAPHICALLY P U R E L U C I F E R I N * K A Z U O H O R I ANn M I L T O N J. C O R M I E R

Deparlment of Chemistry, University of Georgia, Athens, Ga. and University of Georgia, Marine Institute, Sapelo Island, Ga. ( U.S.A .) (Received J a n u a r y 8th, 1965)

SUMMARY

I. The details of a method are presented that describe the isolation of Renilla reniformis luciferin in chromatographically pure form. In addition, results of a study of the absorption and fluorescence characteristics of luciferin are presented. Luciferin absorbs in the ultraviolet at 280 mr* with shoulders at 272 and 288 mr*, but has no absorption in the visible range. 2. Fluorescence excitation and emission spectra of luciferin, at pH 13, show bands at 290 and 362 mt~ respectively, while at p H I the positions of these bands (288 and 355 m/,) are shifted to shorter wavelengths. 3. The shape of the luciferin ultraviolet absorption spectrum indicates a relationship to 2- or 3-substituted indoles. This is also true of the fluorescence data.

INTRODUCTION

Previous studies on this luminescence system have shown that Renilla luciferase catalyzes a reaction between 3',5'-diphosphoadenosine (Ado-3',5'-P2), Ca 2+, Renilla luciferin, and O~ to produce a visible luminescence which has an emission maximum at 485 m/P, 2. It is also known that an easily oxidizable intermediate (activated luciferin) is formed upon (a) incubating luciferase, Ca ~÷, Ado-3',5'-P2, and lucifefin together under anaerobic conditions, or (b) by simply treating an aqueous solution of luciferin at Ioo ° at p H I under anaerobic conditions2, 3. In either case the intermediate formed appears to be the same compound, and the evidence indicates that the activation reaction represents the cleavage of some unknown group from the lueiferin molecule s, 3. These relationships may be illustrated as follows: luciferyl-X luciferin + 0 2

Luciferase + Ca 2+ + Ado-3',5'-P2 ~- l u c i f e r i n Luciferase

luciferin + 0 2

+

X

~ light ~ oxidized luciferin (dark reaction)

* C o n t r i b u t i o n No. 8 4 f r o m the U n i v e r s i t y of Georgia Marine I n s t i t u t e , Sapelo Island, Ga., U.S.A.

Biochim. Biophys. Acta, lO2 (1965) 386-396

CHARACTERISTICS OF RENILLA LUCIFERIN

387

In an effort to understand the mechanisms of the activation step and light reaction, we have concentrated, in part, on the isolation and structural identification of luciferin. We wish to report a method for the isolation of chromatographically pure luciferin and to present some data on the absorption and fluorescence characteristics of this compound. METHODS

Isolation of luciferin Freshly collected organisms were washed in running sea water, the excess water removed by blotting with absorbent paper, and then placed in cold (2~43) saturated ammonium sulfate (pH 7-5). Bottles were filled with cold saturated ammonium sulfate (pH 7.5) three-fourths full and sea pansies added to fill the bottle. The contents were stirred briefly and the organisms allowed to remain in cold saturated ammonium sulfate for 30 min or longer. The animals were removed from the ammonium sulfate solution and put through an electric grinding mill at 2-4 ° . This material was ground further using a Virtis model 45 macro homogenizer for IO rain at high speed. This operation involved placing the material, initially ground by the electric mill, into cold saturated ammonium sulfate (pH 7-5) (three-fourths volume ammonium sulfate to one-fourth of ground organisms) prior to grinding with the Virtis. The homogenized material was centrifuged at 2-4 ° for 15 min at 12000 × g and the supernatant discarded. The precipitate (Renilla residue) m a y be kept at --20 ° for months without significant loss of luciferin or luciferase activity. i kg of Renilla residue was extracted with I 1 of diethyl ether for io min with vigorous mechanical stirring and the ether removed by decantation. This process was repeated. The ether-extracted residue was extracted with I 1 of chloroform in the same manner as above and the chloroform decanted. I 1 of Tris buffer (0.005 M, p H 7.5), preheated to 80 °, was added to the residue and stirred for 15 rain. After centrifugation at 12 ooo :,: g for 15 min the precipitate and supernatant were both extracted for luciferin as follows: (a) The precipitate was extracted with I 1 of ethyl acetate (saturated with 1% Na2COa) at 35-4 °0 for 15 min with vigorous stirring and the solvent removed by decantation. This process was repeated twice. (b) The supernatant was extracted with 800 ml of ethyl acetate (saturated with 1% Na2COa) in a separatory funnel. This step was repeated. The combined ethyl acetate extracts from (a) and (b) above were flash evaporated at 4 °0 using a Buchler evaporator. The yellow oil that remained was dissolved in io ml 95 % ethanol-o.ooi M Tris (pH 7.5)- After storage overnight at --20 ° a precipitate forms which is inactive and can be removed b y centrifugation at o ° for IO min at 12 ooo × g. The supernatant ---- fraction P-I. This fraction contains at least 9 ° % of the detectable luciferin from the Renilla residue, and since Renilla luciferin is quite stable at alkaline pH there is good reason to believe that this method gives good recovery. Ethanolic solutions of luciferin, prepared as above, are stable for at least 3 months at --20 °. Fractions P-l, obtained from IOOOO g of Renilla residue, are further purified as follows: Water is added with stirring to 30 % saturation. The precipitate, which is inactive, was removed by centrifugation for io min at 17000 x g. The supernatant Biochim. Biophys. Acta, lO2 (1965) 3 8 6 - 3 9 6

388

K. HORI, M. J. CORMIER

solution was extracted in a separatory funnel using one-third of a volume of benzene. The benzene e x t r a c t i o n was repeated. A considerable a m o u n t of f a t t y material is r e m o v e d b y this procedure leaving a straw-colored solution of luciferin in the e t h a n o l water layer. The luciferin solution was e v a p o r a t e d to a yellow oil in a Buchler flash e v a p o r a t o r at 35 °. The yellow oil was dissolved in 5 ml of 95 % e t h a n o l - o . o o I M Tris (pH 7-5) a n d e v a p o r a t e d as before. This process was repeated in order to remove the last traces of the benzene. The resulting oil was again dissolved in 5 ml of 95 % e t h a n o l - o . o o I M Tris (pH 7.5) a n d kept at - - 2 0 ° u n d e r N 2. U n d e r these conditions luciferin is stable for m o n t h s . A n inactive precipitate forms which can be r e m o v e d b y centrifugation at o ° for IO m i n at 17 ooo × g. The active s u p e r n a t a n t = fraction W I E . Fraction WIE, prepared from IO kg of Renilla residue, was c o n c e n t r a t e d to a volume of 15-2o ml a n d further purified b y chromatographic m e t h o d s using anaerobic c h r o m a t o g r a p h y on a 3-5 X 30 cm A120 3 column. The A120 a is washed with water, 80 % ethanol, a n d 95 % ethanol, in t h a t order. The A120 3 was equilibrated with 80 % e t h a n o l on the c o l u m n u n d e r a purified N 2 atmosphere. The luciferin fraction was i n t r o d u c e d to the column u n d e r a stream of N 2, e l u t e d w i t h 80 % ethanol, a n d fractions collected anaerobically. The position of the active fraction can be estimated on the c o l u m n b y observing the fluorescence b a n d s using a Keise ultraviolet l a m p with an emission m a x i m u m of 365 m~. The relationship of the color of the fluorescent b a n d s a n d a b s o r b a n c y of the fraction to a c t i v i t y is i n d i c a t e d in Table I a n d Fig. I. The anaerobic column used here is a modified version of the one used to purify Cypridina luciferina, a n d a drawing of our a p p a r a t u s is shown in Fig. 2. TABLE I A B S O R P T I O N , FLUORI~SCENCI~, AND A C T I V I T Y OBSERVATIONS ON FRACTIONS FROM A 1 2 0 3 COLUMN CHROMATOGRAPHY

Fraction

Volume (ml)

i

5

2 3 4 5 6

46 18 2o 25 36

Color

light yellow none none none none none

Fluorescent color

Ultraviolet absorption (m#)

Relative light intensity

Maximum

Minimum

pale blue

28o

275

o

bright blue pale blue quenching zone none none

262 272 272 272 277

25o 242 248 255 257

7oo 16oo 135o 820 400

The two most active fractions (about 35 ml) o b t a i n e d from the A120 3 c o l u m n were c o m b i n e d a n d r e c h r o m a t o g r a p h e d on DEAE-cellulose ( D E A E - T L C cellulose from Gallard-Schlesinger). The DEAE-cellulose was washed with water a n d ethanol following the same procedure used with AlcOa. A 2 :,,~ 5 cm DEAE-cellulose c o l u m n was prepared a n d e q u i l i b r a t e d with 80 % ethanol u n d e r a purified N~ atmosphere. The c o m b i n e d luciferin fractions were i n t r o d u c e d to the column, u n d e r a stream of N~, a n d the column washed with 80 % ethanol u n t i l the fractions showed the absence of 260-m/~ absorbing material. F r a c t i o n s were collected u n d e r anaerobic conditions, a n d a drawing of the a p p a r a t u s used is shown in Fig. 3. The column was t h e n washed with distilled water u n t i l the smell of ethanol could no longer be detected in the Biochim. Biophys. Acta, io2 (1965) 386-396

389

CHARACTERISTICS OF RENILLA LUCIFERIbr

Fig. I. Ultraviolet absorption spectra of fractions from A1203 column chromatography. 150 t

30

75

15

Z 150

==

~

30 15

75 250

300

j

250

300

300 250 WAVELENGTH (m~)

500

I0 I

250

~12 gas

.on t u b e Colle( tq

- N 2 gas

Fig. 2. Column used for anaerobic chromatography on AlzO3. Fig. 3. Column used for anaerobic chromatography on DEAE-cellulose. Biochim. Biophys. Acta, lO2 (1965) 386--396

39 °

K. H O R I , M. J. C O R M I E R

eluate. At this stage, all visible fluorescence is removed from the column. The water fractions were devoid of ultraviolet absorption. Luciferin acts as a strong anion and remains attached to the top of the D E A E cellulose column. Following the water wash luciferin is eluted from the column by washing with Ba(OH)~, pH 12.5. The pH of the fractions is followed, and the fraction eluted between pH 6 to IO routinely contains all of the luciferin activity. The luciferin fraction was kept at --20 ° overnight, centrifuged to remove t3aC03, and flushed with N2-C02 (95:5, v/v) until the pH dropped to 9-9.5. It was stored at --20 ° overnight, TABLE

II

ABSORPTION, FLUORESCENCE, AND ACTIVITY OBSERVATIONS ON I~'RACTIONSFROM I)EAW--CELLULOSE CHROMATOGRAPHY F r a c t i o n s I a n d 2 a r e c o l u m n effluents w h i l e F r a c t i o n s 3 - 5 a r e c o l u m n e l u a t e s u s i n g 8o % e t h a n o l , and Fractions 6-8 represent column eluates using Ba(OH)v

Fraction

I 2 3 4 5 6 7 8

Volume (ml)

Color

5.o 22.o 4.o 4.5 IO.O 0.8 3-5 24.0

none none none none none none none none

Fluorescent color

Ultraviolet absorption (m#) Maximum

pale blue pale blue pale blue bright blue pale blue none none none

Minimum

end absorption 283 243 261 239 269 237 --269 243 28o 251 266 244

Relative light intensity

o o o o o o 380 3°

I0

05

2~

250

300

250

300

250

300

I0

0 03

0.5

25

250

300

I 5

I

I

8

o5~-

250

300 WAVELENGTH

250

300

( mJ.,' )

F i g . 4. U l t r a v i o l e t a b s o r p t i o n s p e c t r a of f r a c t i o n s f r o m D E A E - c e l l u l o s e

Biochim. Biophys. Acta, lO2 (1965) 3 8 6 - 3 9 6

column chromatography.

391

CHARACTERISTICS OF RENILLA LUCIFERIN

centrifuged to remove BaC03, and kept under purified N 2 at --20 °. This solution is referred to as "chromatographically pure luciferin". The correlation between activity and absorption characteristics of the fraction from DEAE-cellutose column chromatography is shown in Table I I and Fig. 4. When this fraction is rechromatographed on DEAE-cellulose thin-layer plates or on paper using these different solvent systems luciferin migrates as a single spot and its absorption characteristics do not change. A schematic diagram illustrating the isolation procedure is shown in Fig. 5. Renilla residue h

Ether

(2 ×) Chloroform

Fat

(i x)

I

Tris buffer, 80 °

Fat

I

I

Buffer

Residue

Ethyl acetate

(2 x)

I

Buffer

Ethyl acetate

(s x)

h

- -R eIs i d u e

I

Ethyl acetate extracts

Ethyl acetate extracts

I

L

Combine, d r y a t 3 °o , dissolve in ethanol P-I Add water (3 ° % b y v o l u m e ) _

_

I

Fat

Luciferin solution Benzene; i ]3 v o l u m e

(2 ×)

I Luciferin s o l u t i o n

I

Benzene layer

D r y , dissolve in ethanol WlE

l I

Alumina column chromatography Active fractions DEAE-cellulose column chromatography C h r o m a t o g r a p h i c a l l y p u r e luciferin Fig. 5. A s c h e m a t i c d i a g r a m of t h e luciferin isolation procedure.

Biochim. B~ophys. Acta, lO2 (1965) 386-396

392

i~. HORI, M. J. CORMIER

Recovery of luciferin Luciferin is quite stable at neutral or alkaline pH values when kept at room temperature or below. It is stable at pH 7-9 for hours at IOO°, and no significant changes occur at pH 13 at this elevated temperature for 5-1o rain. Thus there is good reason to believe that the method given above results in essentially complete recovery up to fraction WIE. A120 3 column chromatography, however, entails using 80 % ethanol as a solvent and a 50 % loss of luciferin occurs possibly due to the acidic nature of luciferin which, when concentrated on the column, results in a significant loss by acid activation 3. In fact, activated luciferin can be detected in fractions preceding the elution of luciferin from the column. Never more than IO % of the luciferin activity is lost by DEAE-cellulose column chromatography. Although 80 % ethanol is also used as an eluent in this step, luciferin is probably protected upon combination with DEAE-cellulose. Thus the overall yield is about 40 %. Sufficient knowledge of the chemical properties of luciferin is now available which should allow us to greatly increase the yields in the future. At the present time approx. 5-1o mg of compound can be obtained from 30000 animals.

Fluorescence measurements These measurements were made using an Aminco-Keirs spectrophosphorimeter, and the values obtained are uncorrected for I P 2 I photomultiplier sensitivity. Aqueous solutions of all compounds examined were used.

A bsorbancy measurements Absorption spectra of the various luciferin fractions were made using a Cary model 14 spectrophotometer.

Preparations of acid-activated luciferin This material was prepared as described previously 3 except that our starting material here was chromatographically pure luciferin. RESULTS AND DISCUSSION

Absorption and fluorescence characteristics of column fractions Table I and Fig. I illustrate the relationship of activity to fluorescence and absorbancy of the fractions from A120 3 column chromatography. The more active fractions usually show little or no fluorescence but a pronounced ultraviolet quenching property. The quenching characteristic of these luciferin fractions is due to a contaminant that has been tentatively identified, by ultraviolet spectroscopy and chromatographic techniques, as deoxyadenosine. We now know that the 272-m/* absorption peak is due to a combination of 26o-m/~ absorption by "deoxyadenosine" and the 280 m/, absorption of luciferin. Table I I and Fig. 4 illustrate fluorescence and absorbancy properties of fractions obtained from DEAE-cellulose column chromatography and their relationship to luciferin activity. "Deoxyadenosine" comes off in the first fraction followed by a blue fluorescent compound. Both of these are eluted from DEAE-cellulose with 8o% ethanol. After washing the column with water, as described under METHODS luciferin Biochim. Biophys. Acta, lO2 (1965) 386 396

CHARACTERISTICS

393

OF R E N I L L A L U C I F E R I N

is eluted from the column with Ba(OH) 2. The second Ba(OH) 2 fraction contains 9 ° % or more of the luciferin activity put on the DEAE-cellulose column. Absorbancy from 250-35o m/~ is negligible in fractions after No. 8. As indicated under METHODS this luciferin preparation appears to be chromatographically pure.

Absorption spectrum of luciferin Fig. 6 illustrates the absorption spectrum of Renilla luciferin. In the ultraviolet there exist one major absorption band at 280 m/~ with a shoulder at 272 m/, and another at 288 m/~. There is no absorption in the visible region of the spectrum. The observed absorption characteristics of luciferin closely resemble that of the 2- or 3substituted indoles. For comparative purposes the absorption spectrum of indican is shown here. The close similarities in the absorption characteristics of the two compounds are evident. 40

E--

1.0

i

~LUCIFERIN (pH7}

LUCIFERIN-- I 0.8

0.8

0.6

0.6

AClD ACT/VAT~Ol) ~o 03 Q3

< 0.4

0.4

0.2

3.2

i

210

i

240 WAVELENGTH

270

i

i

300

350

i

(rnp)

i

210

i

r

240

270

WAVELENGTH

i

i

300

330

(m a)

Fig. 6, Ultraviolet absorption spectra of luciferin and a comparison to t h a t of indican. Fig. 7. U l t r a v i o l e t a b s o r p t i o n s p e c t r a of a c i d - a c t i v a t e d l u c i f e r i n a t a c i d a n d a l k a l i n e p H .

A bsorption spectrum of activated luciferin Fig. 7 illustrates the absorption spectrum of acid-activated luciferin at acid and alkaline pH. The absorption spectrum of luciferin is included for comparative purposes. It is evident that, whatever changes take place during acid activation, this has little effect on the ultraviolet absorption spectrum. There is a I-2-m/~ shift to shorter wavelength with a new absorption shoulder appearing near 330 m/~ under alkaline conditions. Biochim. Biophys. Acta, lO2 (1965) 3 8 6 - 3 9 6

394

i<. HORI, M. J. CORMIER

Fluorescence analysis of luciferin and activated luciferin The fluorescence excitation and emission spectra of luciferin at acid and alkaline pH are shown in Fig. 8. At pH 13 the excitation and emission maxima are at 290 and 362 m/z, respectively, whereas at pH I the position of these maxima (288 and 355 mt~) are shifted to shorter wavelengths. The ultraviolet fluorescence of Renilla luciferin is consistent with the fact that little or no visible fluorescence is associated with the most active column fractions. In addition, the shapes of the fluorescence excitation and emission spectra of luciferin indicate the lack of fluorescent contaminates in our preparation. >I--

].

LUCIFERIN

~4

362

290

Z I.u IZ

--r t

28;

z o~ la.I 0 :) .J la.

3OO

4OO WAVELENGTH

50O

600

(m.,U)

Fig. 8. F l u o r e s c e n c e e x c i t a t i o n a n d emission s p e c t r a of luciferin a t acid a n d alkaline p H .

The shape of the absorption spectra of luciferin indicates a possible relationship to indole-type compounds. This is also true of the fluorescence data. For comparative purposes the fluorescence excitation and emission spectra for indican is shown in Fig. 9. The excitation and emission maxima for this compound are located at 29o and 388 m/z, respectively, thus being quite similar to the spectra obtained for luciferin. INDICAN >..

5

03 Z ea It

4

I--

Z o w

u.,

i

30o

400 WAVELENGTH

50o

600

(m~)

Fig. 9. F l u o r e s c e n c e e x c i t a t i o n a n d e m i s s i o n s p e c t r a of indican.

If a solution of luciferin is acidified with HC1, under anaerobic conditions, and subsequently exposed to air and its fluorescence characteristics studied, the results Biochim. Biophys. Acta, lO2 (1965) 386-396

395

CHARACTERISTICS OF RENILLA LUCIFERIN

shown in Fig. IO are obtained. Although the position of m a x i m u m emission does not change with time, there has always been observed a slight change i n intensity of fluorescence which lasts for about 6 rain. Whereas the intensity of the excitation fluorescence increases during the first few minutes, that of the emission fluorescence decreases. After 6 min a 3o-sec flush with 0 2 stabilizes the excitation and emission intensities. These transient changes in fluorescence intensity that occur, for the most part during the first 6 min, are consistent with our previous measurements on the half-life of acid-activated luciferin when exposed to air s. These changes probably represent the formation of dehydroluciferin via a dark reaction. After autooxidation

>.

~it)

4

Z uJ

.

.

.

.

P-

z

:3

.........

355

288

.

.

.

.

.

.

.

.

.

.

.

.

.

LUCIFERIN, p H I (0 time) 2 minutes 4 minutes

LIJ f.) Z

uJ 2

".~,~" f/.: ~.. '~ ~.~,

-

-

Flush w i t h 02 f o r 30 seconds

w 0

I

_1 LL

300

400

500

600

WAVELENGTH (re.e)

Fig. io. T r a n s i e n t c h a n g e s t h a t occur in t h e fluorescence e x c i t a t i o n a n d emission s p e c t r a of luciferin following acid a c t i v a t i o n a n d e x p o s u r e to 02 of t h e a t m o s p h e r e . i

f

A C I D ACTIVATED L U C I F E R I N )t,,- 5 ~:~

z_

i

288 362

4

Z

i

AFTER A U T O O X I D A T I O N

~pH

5~

!88

#2

300

400 WAVELENGTH

F i g . I I. F l u o r e s c e n c e

500

600

{re.a)

excitation and emission spectra of autooxidized

acid-activated

luciferin at

acid a n d alkaline p H .

of acid-activated luciferin is complete, the results shown in Fig. i i are obtained. Fig. II illustrates the fluorescence excitation and emission spectra of autooxidized acidactivated luciferin at acid and alkaline pH values. The close similarity of these Biochim. Biophys. Acta, Io2 (1965) 3 8 6 - 3 9 6

39 0

K. HORI, M . J . CORMIER

s p e c t r a w i t h those o b t a i n e d using luciferin in Fig. 8, indicates t h a t v e r y little change in s t r u c t u r e h a s occurred t h a t w o u l d h a v e a n y effect on the fluorescence characteristics of luciferin. Similarities in the a b s o r p t i o n s p e c t r a of luciferin a n d a c i d - a c t i v a t e d luciferin also p o i n t to small changes in s t r u c t u r e a c c o m p a n y i n g the a c t i v a t i o n process. One of t h e i n t e r e s t i n g i m p l i c a t i o n s of these studies is t h a t R e n i l l a luciferin m a y be an indole d e r i v a t i v e . A s t u d y of its chemical p r o p e r t i e s also p o i n t s to an indole, a n d t h e results of these i n v e s t i g a t i o n s will be p u b l i s h e d elsewhere. I n this connection it is of i n t e r e s t t h a t crystalline C y p r i d i n a luciferin, after reduction, is c o n v e r t e d to a c o m p o u n d which, according to its a b s o r p t i o n s p e c t r a a n d chemical characteristics, also a p p e a r s to be an indole d e r i v a t i v e 5. I n addition, we h a v e r e c e n t l y r e p o r t e d the fact t h a t indole a n d its 3 - s u b s t i t u t e d d e r i v a t i v e s chemiluminesce at alkaline p H w i t h a s p e c t r a l energy d i s t r i b u t i o n v e r y close to t h a t of R. reniformis bioluminescence 2. ACKNOWLEDGEMENTS

This w o r k was s u p p o r t e d in p a r t b y t h e N a t i o n a l Science F o u n d a t i o n a n d the U.S. A t o m i c E n e r g y Commission. T h e a u t h o r s w o u l d also like to acknowledge t h e c o m p e t e n t t e c h n i c a l assistance of Mrs. L. B. HAWLEY a n d Mr. PHIL PI~ICI~ARD as well a s the facilities p r o v i d e d b y t h e Sapelo R e s e a r c h F o u n d a t i o n . W e are also grateful to C a p t a i n F. TODD a n d t h e Valona, Georgia s h r i m p e r s who h e l p e d to organize a n d collect m a s s i v e a m o u n t s of sea pansies used in this work. REFERENCES i M. J. CORMIER,J. Biol. Chem., 237 (1962) 2o32. 2 IV[.J. CORMIERAND C. B. ECKROADE, Biochim. Biophys. Acta, 64 (1962) 34o. 3 M. J. CORMIERANI) K. HORI, Biochim. Biophys. Acta, 88 (1964) 994 0 . SHIMOMURA,T. GOTO AND Y. HIRATA, Bull. Chem. Soc. Japan, 3° (1957) 929. .5 Y. ]-IIRATA,O. SHIMOMURAAND S. EGUCHI, Tetrahedron Letters, 5 (1959) 4Biochim. Biophys. Acta, lO2 (1965) 386-396