Bioluminescent particles from the marine dinoflagellate ...€¦ · dinoflagellates (3, 11, 12) and in other systems, notably the firefly (17). Knowl- edge of the physiological aspects

The Characterization of Scintillons

Bioluminescent particles from the marine dinoflagellate, Gonyaulax polyedra

R I C H A R D DESA and J. W O O D L A N D H A S T I N G S

From the Division of Biochemistry, University of Illinois, Urbana, Illinois 61801, and the Marine Biological Laboratory, Woods Hole, Massachusetts 02543. Dr. DeSa's present address is Biochemistry Department, Wing Hall, Cornell University, Ithaca, New York 14850. Dr. Hastings's present address is The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

ABSTRACT A new type of biological particle, isolated from the marine dinoflagellate Gonyaulax polyedra, has been partially purified and characterized. When the pH is lowered, the particle emits light in vitro in a fashion closely mimicking the flash of the living cell, and it is referred to as a scintillon (flashing unit). Scintillons are obtained by breaking the cells in buffer at pH 8.2 and purifying by differential and sucrose density gradient centrifugation. The particle has a density of about 1.23 g cc -1, and activity is quantitatively correlated with the number of crystal-like birhombohedral structures. These have been found to contain guanine, but since the density of authentic guanine is about 1.73 g cc -1, the scintillon is believed to comprise additional but as yet unidentified components. The properties of the scintillon and the effects of various physical and chemical treatments are described. The reasons for believing that this particle is responsible for the flash of the intact cell are discussed.

I N T R O D U C T I O N

Bioluminescence in mar ine dinoflagellates such as Gonyaulax or Noctiluca occurs as a brief, br ight flash (10, 12) upon s t imulat ion of the organism. T h e subcel lular o rganiza t ion and contro l mechanisms involved in such flashing con- sti tute an interest ing bu t little invest igated p rob lem in cell biology. Bio- chemica l studies of the componen t s involved have been car r ied out bo th in dinoflagellates (3, 11, 12) and in o ther systems, no t ab ly the firefly (17). K n o w l - edge of the physiological aspects of flashing comes f rom several studies, par t icu la r ly those of Buck et al. (4) on fireflies, and the recent work of Ecker t on Noctiluca (7, 8).

Ev idence concern ing the na tu re of a subcel lular b io luminescent system a t a level of organiza t ion above the molecu la r was first ob ta ined in studies wi th

m5

The Journal of General Physiology

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Published January 1, 1968

~o6 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y • V O L U M E 5 1 • 1968

Gonyaulax (5, 6). I t was found tha t a par t ic le could be isolated at p H 8.2 which, when the p H was lowered to 5.7, would emi t a flash very similar to tha t of the living cell. T h e t e rm scintillon (flashing unit) was coined to refer to this particle. I t was j u d g e d tha t the scintillon was involved in the flashing of the in tac t organism, p re sumab ly being appropr i a t e ly e m b e d d e d in the cy top lasm and control led by m e m b r a n e - r e l a t e d electrophysiological events, such as have been recen t ly e luc ida ted by Ecker t in Noctiluca (7, 8).

This pape r describes studies conce rned with the propert ies and ident i ty of scintillons. T h e re la t ionship be tween scintillons and the previously descr ibed soluble enzyme and substrate which results in b ioluminescence was considered in a recent review article in a sympos ium vo lume (13), where some addi t ional exper iments conce rned wi th scintillons were also presented.

M A T E R I A L S AND M E T H O D S

The photosynthetic marine dinoflagellate Gonyaulax polyedra was grown in a supple- mented seawater medium (93) using fluorescent lamps scheduled to provide alternate 12 hr periods of light and dark. Cells were harvested by filtration during the light period, at which time the best yields of scintillons were obtained.

Active scintillons were isolated by relatively gende mechanical rupture of the cells in buffer at pH 8.2 using a TenBroeck homogenizer (13). Large debris such as un- broken cells, cell walls, etc. was removed by low-speed centrifugation; active particles can then be sedimented at 30,000 g for 10 rain. The resuspended pellet provides a source of crude scintillons.

Purification was carried out by isopycnic density gradient centrifugation using either the Servall refrigerated or the Spinco L-2 centrifuge. Gradients were prepared by layering different concentrations of sucrose usually ranging from 1.08 to 1.32 g cc -1. In this technique a particle comes to rest in the tube at a position corresponding simply to its buoyant density. Scintillons have a density of about 1.23 g cc -l, and are relatively large. They move to a position close to the equilibrium relatively rapidly. At 25,000 rpm in the SW 25.1 rotor a discrete band is well-formed within 45 rain; between 2 and 5 hr are required for final equilibrium to be achieved, which involves primarily a narrowing of the half-band width (24).

Bioluminescence was measured utilizing a photomultiplier, recording with either an oscilloscope or a high-speed graphic recorder. For experiments in which prompt and uniform mixing was required, the stopped-flow rapid mixing apparatus of Gibson and Milnes (9) was used. In routine assays mixing was achieved simply by rapidly squirting 0.03 N acetic acid from a syringe into a vial containing scintillons positioned above the phototube. In both cases, equal volumes of the two solutions were used, the scintillons being suspended in 0.05 M Tris buffer, pH 8.2 with 4 % sucrose added. The sucrose was found not to be essential and was omitted in some of the later work. The pH of all buffers was measured at room temperature even though many of the experiments presented were carried out at lower temperatures.

l~luorescence and phosphorescence measurements were made with the Aminco- Bowman 4-8100 spectrophotofluorometer and absorption measurements with the

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RICHARD DESA AND J. WOODLAND HASTINGS Characterization of Scintillons Io 7

Cary Model 15, the Bausch and Lomb 505, or Zeiss PMQ, II spectrophotometer. Electron microscopy was performed using standard techniques with the RCA EMU-3 microscope; 50 Kv accelerating voltage was used throughout.

R E S U L T S

If scintillons collected only from the center of the active region in a sucrose density gradient are layered again on another gradient, a second sedimentation results in a purification such that the bulk of the visible particulate impu- rities is separated from the activity. Inspection by electron microscopy of the fractions collected from this centrifugation reveals the presence of crystalline- like structures as the major component (Fig. 1). Their numbers, as measured by the sprayed droplet technique of Backus and Williams (1), are very closely proportional to the scintillon activity measured in samples from the same fractions (Fig. 2). Although other materials are present in the electron microscope pictures, no other structure is seen to occur predominantly in the tubes having scintillon activity.

As may be noted from Fig. 1, the size of individual particles varies sub- stantially, but there appears to be no correlation between their distribution in the density gradient and size.

If, instead of using the more purified preparations from the second gradient, one examines in the electron microscope the fractions from a first gradient, one finds that there are many more of these crystal-like structures, especially at the bot tom of the tube. However, the activity is still found exclusively at the characteristic level. The quanti ty of this heavier crystalline material is very substantial, so that its isolation in quanti ty is not difficult. Suspended in H 2 0 at neutral pH, the crystals exhibit an absorption (Fig. 3) which may be largely attr ibuted to Rayleigh scattering, although an anomalous and as yet unexplained absorption band occurs at about 325 m~. The crystals are soluble in dilute acid or base and the absorption in the ultraviolet is then very apparent, being specifically identifiable as that of guanine (Fig. 4).

The distribution of the crystals after centrifugation of the crude scintillon preparation in a density gradient may be deduced from the measurements shown in Fig. 5, where the location of the guanine is estimated by its fluorescence. Also shown in this figure is the distribution of scintillon activity and that of visible absorption, measured at 675 m~, and assumed to be due primarily to chloroplast fragments.

Although guanine is apparently not known to crystallize in the birhombohedral form, our identification of the crystals as guanine seems unambiguous. In addition to the absorption spectra, which are highly diagnostic, the fluorescence and phosphorescence match those of authentic guanine (Fig. 4), including the lifetime of the phosphorescence (tl/~ = 1.41 see) at 77°K. In addition, although not illustrated in the figure, the guanine isolated from the

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FIGURE 1 a and b

io8

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RICHARD DESA AND J . WOODLAND HASTINGS Characterization of Scintillons I o 9

FIGURE 1 C

FIGURE 1. Examples of the electron micrographs used in obtaining the data presented in Fig. 9. Fractions collected from a sucrose density gradient centrifugation were mixed with a known number of polystyrene latex particles (288 m# diameter) and the mix- tures were then sprayed onto microscope grids according to the method of Backus and Williams (1). Electron micrographs were then taken of droplets on the several grids. An average of 19 droplets was photographed from each fraction and a count was made of the number of scintillons and latex particles present. The data plotted in Fig. 2 are an average of all the droplets counted for a given fraction. Figs. 1 a, 1 b, and 1 c are photographs of samples from fractions 8 9, 10, and 11 respectively of Fig. 2. S = scintil- Ions; L = latex particles

cel ls e x h i b i t s t h e c h a r a c t e r i s t i c s p e c t r a l shifts w h i c h o c c u r u p o n c h a n g i n g the

p H (16).

Ce l l g u a n i n e a n d a u t h e n t i c g u a n i n e r a n t o g e t h e r o n p a p e r c h r o m a t o g r a m s

in t w o d i f f e r e n t s o l v e n t sys t ems a n d n o o t h e r u l t r a v i o l e t - a b s o r b i n g spo ts w e r e

d e t e c t e d on the d e v e l o p e d c h r o m a t o g r a m s . I n a b u t a n o l - w a t e r s o l v e n t a n

R i of 0 .15 was o b t a i n e d for b o t h s ample s . I n a w a t e r s y s t e m ( p H a d j u s t e d to

10 w i t h N H 4 O H ) a u t h e n t i c g u a n i n e m o v e d 15.4 c m c o m p a r e d to 15.6 c m

for cel l g u a n i n e . T h e R I v a l u e s w e r e n o t d e t e r m i n e d in this case b e c a u s e to

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140

THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 51 • 1968

120

o I00

£

'___ 80

x

< 6 0 I..-.- z ,<

o 40

20

I 2 4 6 8 I0

FRACTION NUMBER

I I O

180

~2 - 1 6 0 v

- 1 4 0 7 0

- 1 2 0 ×

r r -

- - I00 w (13

- - 8 0

60 "' J

40 < D._

20 x

0 12 14

FIGURE 2. Sedimentation pattern of bioluminescent particles following centrifugation in a sucrose density gradient. 5 ml of a particle suspension were layered on a linear sucrose gradient (1.08 to 1.32 g cc -1) and centrifuged at 25,000 rpm in the Spinco SW 25.1 for 45 min at 0°C. This time is adequate for the scintillons to band close to the final equilib- r ium position (24). Bioluminescence is correlated with the presence of chevron-shaped particles. The ordinates are, left, bioluminescence, expressed as the total number of quanta emitted by the fraction in the standard scintillon assay; right, total number of chevron-shaped particles in the fraction.

12 F [CRYSTALS DISSOLVED l

06 CRYSTALS H20 CRYSTALS SUSPENDED

° 2 - 1 I I I I ~ 1 I [ [ I 220 240 260 280 300 320 340 360 380 4.00

WAVELENGTH, m u

FIGURE 3. This figure presents the absorption spectra of the chevron-shaped crystals isolated from the bottom fractions of a sucrose density gradient centrifugation experiment (Fig. 5). These crystals were simply resuspended in water to obtain the spectrum labeled "crystals suspended in H20;" examination of a similar quanti ty of crystals dissolved in 0.1 N HC1 produced the other spectrum and indicates that the substance was guanine.

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RICHARD mESA AND J. WOODLAND HASTINGS Characterization o/Scintillons I I I

improve resolution the solvent front was allowed to move off the paper, which was 39 cm long.

The buoyant density of both authentic and cell guanine crystals was found to be between 1.73 and 1.74 g cc -1. The determination was made in the Spinco SW39 rotor utilizing a preformed gradient of CsC1; 0.05 ml of dilute suspension of the guanine crystals was layered on top of the gradient and centrifuged at 105,000 g for 1 hr. Fractions were then obtained by piercing the bottom of the centrifuge tube with a needle and collecting drops. Guanine was esti-

I-~l / '~ ABSORPTION 2oH~ / k.

F\d -h F--AUTHENT'C GUAN'NEI IOL !1,7 ~ .... CELL GUANINE

~ o' I r , , / , I I I i ~n - ACTIVATION ~ ~EMISSION FLUORESCENCE " A / \ 7

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'<~°r / / \',,, / \ , AT77OK b / I \ ' - . if "%.

5ol- /I \ ' - . . /' \ ~/ ".

0 30O 4OO 500 WAVELENGTH

FIOURE 4. This figure illustrates the results of a comparison between authentic guanine and guanine isolated in the form of crystals from Gonyaulax. The absorption spectra were obtained by dissolving the samples in 0.1 N HC1. Fluorescence was measured on samples dissolved in 0. I N I-IC1 and held at 77°K in liquid nitrogen. The fluorescence and phosphorescence data are uncorrected for wavelength calibration, monochromator efficiency, and phototube sensitivity.

mated from the optical density at 275 m# of aliquots diluted in 0.1 N HC1. The density of each fraction was then determined accurately by measuring the refractive index in a calibrated Zeiss refractometer and converting these data to density values with the relationship given by Ifft et al. (15). Four experiments were performed, giving values of 1.739, 1.728, 1.741, and 1.731 g cc -I, the first two for authentic guanine and the latter two for cell guanine. The actual value for the density cannot be assigned more accurately because of the limitations imposed by the method of collecting fractions. More accurate determinations could be made with the analytical centrifuge.

The crystals (Fig. 1) which correlate in number with scintillon activity

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I I 2 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y • V O L U M E 5 1 • I 9 6 8

(Fig. 2) appear identical in the electron microscope wi th those cell guan ine crystals wh ich occur a b u n d a n t l y in the pellet fraction during purif ication (6, 13) and fluorescence measurements of al iquots from the scinti l lon band indicate the presence of guanine (Fig. 5, and Findlay, unpubl i shed data) .

po 7--° 8 I BIOLUMIN e ~

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This figure illustrates the distribution of particles of several types after centrifugation of a crude scintillon preparation in a sucrose density gradient. 5 ml of a particle suspension were layered on a linear sucrose gradient (1.08 to 1.32 g cc -I) and centrifuged at 25,000 rpm in the Spinco SW 25 for 30 rain at 0°C. Fractions were then obtained by piercing the bottom of the centrifuge tube w i t h a needle and collecting drops. Scintillons were measured by their bioluminescence; colored material (primarily chlorophyll) was estimated from the absorption of the fraction at 675 m#; and guanine was measured by diluting an aliquot of each fraction with 1 N NH4OH and then measuring the fluorescence at 360 In# upon excitation at 275 m#. Sedimentation was from left to right.

These observations suggest that the scinti l lon is a complex structure having a guan ine crystal associated wi th it in some way. Nei ther authent ic guan ine nor cell guan ine has b io luminescence activity, nor does added guan ine in any w a y inf luence the activity or stabi l i ty (see be low) of scintillons.

L ight emission has been obta ined from isolated scintiUons on ly by lowering the p H of the suspending medium. Attempts to obtain luminescence in other

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RICHARD DESA AND J. WOOl)LAND HAStinGS Characterization of Scintillons I t 3

ways have not been successful. Quanti tat ive data relating both the maximum initial light intensity and total yield of photons to the final pH are shown in Fig. 6; a relatively sharp opt imum occurs at pH 5.7. The glow which occurs at a low pH has not been studied.

Aside from oxygen, no other factors are required in the reaction. This is illustrated by the experiment of Fig. 7, which shows that the luminescence obtained from a particle preparation is directly proportional to the number of particles over a 100,000-fold range. The results are consistent with the assumption that the activity of each scintillon is independent of the others, requiring no soluble factor (other than oxygen), and does not involve a factor

I I I I I I 0 0 "¢---4 = I0 ~ QUANTA TOTAL LIGHT (=) ~

"~'---5 x I0 II QUANTA SEC-'- INTENSITY ( o ) d f / ~ 9"c / / tX

e / i /\ 6 0 - -

4 0 - -

li- t, i :~ 2 0 - - ",,,,.

2 3 4 5 6 7

FINAL pH

Fmum~ 6. The effect of pH on the intensity of light and tota l light produced by isolated scintillons. Measurements were made in the stopped-flow apparatus. Equal volumes of a scintillon suspension at pH 7.6 and buffer at a lower pH were rapidly mixed to arrive at the final pH indicated. The particles were suspended in 4% sucrose, 0.01 M EDTA, buffered to pH 7.6 with 0.005 M Tris-maleate. The buffers used to change the pH were 0.05 M and were selected on the basis of their useful pH ranges.

which is dissociable upon dilution. The deviation at very high concentrations is attributed to self-absorption of light by the turbid, colored materials present in the crude scintillon preparations (see Fig. 5), and not to Lnhibition of activity per se. The fact that the intensity and yield parallel one another documents the observation that the dme course of the flash is independent of dilution.

Oxygen is not required at the moment when the pH is changed, but-will stimulate luminescence if added a few seconds after anaerobic mixLng of acid and scintillons. This matter has not been studied further; the lffedme of the intermediate is not known.

The in vitro flash is temperature-dependent Ln the usual way, the reacdon being faster at higher temperatures over the range of 2 ° to 20°C. Examples

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II4 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 51 • 1968

of the in vi t ro flash a t two tempera tures are shown in Fig. 8. T h e more exten- sive da ta presented in T a b l e I i l lustrate the fact tha t the yield of the react ion is essentially i ndependen t of t empera ture . These da ta also indicate tha t the act ivat ion energy for the onset of luminescence is qui te close to tha t for the decay phase; the la t ter has been es t imated at abou t 11.2 kcal per mole. T h e

to ' "

L INTENSITY 1011 QUANTA SEC -1

10 m

TOTAL LIGHT QUANTA

109 ~ x / / / ~ / ---

10e /

x t0 7 10-5 t0-4 i0-3 10-2 10 -1 10 °

ML PARTICLES

F:oum~ 7. This figure illustrates the linearity between light production and particle number. Each experimental point was obtained by rapidly squirting 1 ml of 0.03 N acetic acid into a vial containing 0.9 ml 0.005 N Tris-maleate buffer, pH 7.6, and 0.1 ml of appropriately diluted particle suspension. Intensity values were read directly from high-speed recorder records and total light values obtained by integration. Assays were performed at approximately 22°C. The lines are theoretical for strict proportionality.

effect of t empera tu re upon the decay phase of the flash, bo th in vivo and in vitro, is i l lustrated in Fig. 9; the a p p a r e n t ac t ivat ion energy differs slightly for the two.

In cont ras t to part icles such as mi tochondr i a or lysosomes, the tonic i ty of the suspending m e d i u m appears to have relat ively little effect upon the act ivi ty of scintillons. So long as the p H is ma in t a ined a t a b o u t p H 8.2, the scintillons appea r to be as stable in distilled wate r as in sa tura ted sucrose or 0.01 N NaC1.

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RICHARD DESA AND J. WOODLAND HAsrmas Characterization of Sdntillons I 15

Some inactivation, which is at least partially reversible, occurs at higher salt concentrations, but this has not been investigated.

Scintillons are subject to physical damage, for example by mechanical grinding or ultrasonic treatment. An activity loss which may also be ascribed to physical damage occurs upon freezing and thawing.

At every temperature investigated thus far, down to 77°K, there is a detect- able loss of activity upon storage. The reason for this is not known, but it is

: ' ° - r ' o _

°

0 I00 200 500

MILLISECONDS

FIOURI~ 8. This figure presents a comparison between the flash produced by scintillons at two different temperatures. Each record was obtained by mixing, in the stopped-flow apparatus, equal volumes of 0.06 N acetic acid and scintillons suspended in buffered sucrose, pH 8.2.

T A B L E I

EFFECT OF T E M P E R A T U R E ON THE IN V I T R O FLASH OF SCINTILLONS

Temperature k, decay, sec-l Io, quanta sec~l X llkal Total quanta X 10-10

°c

2 8.7 3.2 6.1 9 14.5 4.7 5.4

14 18.4 6.9 5.7 18.5 22.4 7.3 5.5 20 25.8 11 6.5

The experiments were carried out as described in the legend to Fig. 8.

temperature-dependent, so that the half-life at 77°K may be measured in weeks or months (24).

Thermal destruction also occurs; scintillons are rapidly inactivated at temperatures above 30°C with a high activation energy, about 82 kcal (Fig. 10). This result indicates that protein may be an essential component of the system.

I t was shown by Hastings and Sweeney (12) that the soluble luminescent system from Gonyaulax requires a high salt concentration for maximum activ-

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xx6 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 5I • x968

[-- iX'* i SCINTILLONS x { - -

~ X 20 ~ # = 11.2 KCAL

_

I0- - ~=19.2 KCAL ~X ~ '

8 - \ \ "

6 - 1 I E i \ l / 3.40 3.50 360

M to 3 y x

FXGURE 9. This figure illustrates the effect of temperature on the rate of decay of luminescence of scintillons and of living cells. The data for scintillons were collected from experiments with the stopped-flow apparatus carried out as described in the legend to Fig. 8. The data for the living cells were obtained by recording the flash elicited from cells after placing them in a vial in front of the phototube and then striking the phototube housing.

1.00

0.50

Z

t 0.I0 v

0.05

I 3.15

CAL

I I k 3.20 3.25 3.30

I x 103

FIGURE 10. This figure illustrates the effect of temperature on the rate of loss of scintil- Ion activity. Aliquots of a scintillon preparation were incubated in the 4% sucrose buffer, at four different temperatures. Samples were removed at timed intervals and assayed for scintillon activity. The first order rate constant, k, was obtained from the slope of the straight line obtained by plotting log activity vs. time. The data are plotted according to the Arrhenius equation and indicate an apparent activation energy for the inactivation process of 82 kcal per mole.

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RICHARD DESA AND J. WOODLAND HASTINGS Characterization of Sdntillons I x7

ity. Hastings and Bode (11) tested a wide variety of salts and found the require- ment to be rather nonspecific and proposed that the effect might involve an effect upon the conformation of luciferase such that the quantum yield of the reaction was increased. It was thus of interest to examine various salts with regard to their effect upon the scintillon system. Some of the results are sum-

T A B L E I I

E F F E C T O F S A L T S O N S C I N T I L L O N B I O L U M I N E S C E N C E

Salt Final concentration Inhibition of I.

moles]liter %

CuC12 1 X 10- s 85 CuC1 2 X 10- 3 80 AgNoa 5 X 10- 5 98 HgC12 1 X 10- 5 90 Z n C l s 1 X 10 -4 75

I n h i b i t i o n of sc in t i l lon b i o l u m i n e s c e n c e by salts. Sc in t i l lons were i n c u b a t e d in E D T A - f r e e buf fe r w i t h sal ts a t t he c o n c e n t r a t i o n s no ted , a n d b io lumines - cencc was i n i t i a t ed by m i x i n g w i t h a n equa l v o l u m e of 0.03 u acet ic acid. A t a c o n c e n t r a t i o n of 10 - s xt sal t , t h e r e was no inh ib i t i on obse rved w i t h the fo l lowing: CsC1, M g C 1 2 , LiC1, KC1, NaC1, BaC12, A I C l s , CrCl s , M n C I ~ . F e C l s , CoCl2 , NiCI2 .

T A B L E I I I

E F F E C T O F Z I N C I O N O N T H E I N T E N S I T Y A N D Q U A N T U M Y I E L D O F S C I N T I L L O N B I O L U M I N E S C E N C E

Zinc concentration Rise time 1Io, quanta so= X 10-u k, decay Total quanta X I0-te

M Sea Sec - I

0 0 .08 6 .6 11.1 12.3 2 X 1 0 - 6 0 .09 3 .7 10.7 7 .8 1X10 -5 0.21 1.0 4 .56 3 .9 5X10 -5 0 .35 0 .59 2 .0 4 .8 1X10- 4 0 .42 0 .42 1.8 4 .5 5X10 -4 0 .65 0 .18 1.1 2 .5 5 X 1 0 - s 0 .90 0 .09 0 .29 2 .4

Sc in t i l lons were i n c u b a t e d for 4 ra in in E D T A - f r e e buf fe r a t t he z inc c o n c e n t r a t i o n s ind ica ted , a n d t h e n m i x e d in t he s topped-f low a p p a r a t u s w i t h a n equa l v o l u m e of 0.03 N acetic ac id to in i t i a te b io luminescence .

marized in Table II. A striking inhibitory effect was observed with salts of silver and mercury at concentrations of about 10 -5 M. The action of copper ions, like that of silver and mercury, decreased the intensity without any effect upon the kinetics, as though a certain fraction of the scintillons was inactivated. With zinc, on the other hand, an inhibition of the bioluminescence intensity was accompanied by a flash having longer duration, such that

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I I 8 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y • V O L U M E 5 I o I968

the final yield was not decreased as much as the initial intensity (Table III) . The mechanism of inhibition by these several ions is not known, but more detailed investigation of the mode of action, especially that of zinc, might be useful. In all cases tested the inhibition could be reversed by t reatment with ethylenediaminetetraacetic acid (EDTA).

A variety of detergents were tested for their action on scintillons. This was of interest in relation to the hypothesis that a lipid-like material might be involved in the scintillon structure, and most of the detergents tried resulted in a complete loss of activity at a concentration of 0.5%. These included Triton X-100, Tri ton W-30, Tri ton 770, Duponal C, and deoxycholate. At the same concentration, however, Tween 80 was without effect upon bioluminescence.

D I S C U S S I O N

These results may be considered in the context of two statements which sum- marize our knowledge and hypotheses concerning scintillons.

1. A large, rapidly sedimenting particle, termed the scintillon, which is capable of emitting a light flash whenever the pH is lowered from about 8 to 5.7, may be isolated and purified from cell extracts. It is hypothesized that this particle exists in a similar form in vivo, presumably embedded in the cytoplasm in an appropriate fashion, and that it is responsible for the luminescent flash of the living cell.

2. I t has been found that the scintillon is associated with chevron-shaped crystalline-like particles, identified as guanine. It is hypothesized that the in vivo functional particle comprises additional, but as yet unidentified components.

In considering these points in turn, there is no question concerning the particulate nature of the scintillon, since it may be repeatedly diluted or sedimented without loss of activity due to these treatments.

The accompanying hypothesis is difficult to prove unequivocally, but the evidence at hand is certainly very strong in its favor. The fact that the in vitro emission closely resembles the flash of the living cell is very suggestive; both the color and the time course (which lasts a fraction of a second) are very similar (13). This would scarcely be expected if the particle arose as an artifact during the extraction procedure. Moreover, the fact that the isolated particle can be manipulated and purified while retaining its specific kinetic and other properties, suggests that it exists as a discrete biological entity. Observations on living cells are even more indicative, for both Gonyaulax and other dinoflagellates are known to emit flashes from discrete spots in the cell (8, 19, 20). If a living cell is crushed between a slide and cover slip so that the cytoplasm simply flows out, light flashes come from discrete spots when the pH is lowered. I t is assumed that these structures correspond on the one

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RICHARD DI~,SA AND J. WOODLAND HASTINGS Characterization of Scintillons I 19

hand to those which were evident in the living cell, and on the other hand to the scinfillons which can be purified from this cytoplasm and respond sim- ilarly to acid.

The second point has been more difficult to establish and less compelling to accept, for it is not immediately evident why or how a guanine crystal might be associated with a light-emitting particle. The conclusion rests primarily upon the experiment of Fig. 2, in which a correlation was found between the number of chevron-shaped particles and scintillon activity. A different experiment providing the same conclusion was reported in the publication which first described scintillons (6). Although that experiment was similar, it was distinctly different because the separation method involved sedimentation for 2 hr through a 5-20°7o sucrose gradient at only 3200 rpm in the International PR-1 centrifuge with the swinging bucket rotor. Tha t procedure, therefore, involved the sedimentation velocity of scintillons, where- as the present result relates to their buoyant denstiy. The scintillon activity is thus correlated with the number of chevron-shaped particles following purification by either of two distinctly different methods.

On some occasions it was found that the peak activity did not correspond exactly to the distribution of the crystals in experiments similar to that of Fig. 2 (13). This is now believed to be due to the fact that spontaneously inactivated scintillons become slightly more dense (Hastings and Vergin, unpublished results), so that the apparent distribution of chevron-shaped particles may not correlate exactly with activity when a mixture of active and in- active particles is present.

The disparity in densities between the chevron-shaped guanine crystals from the cell and those from the scintillons requires explanation. If the guanine crystal is indeed associated with the active scintillon, as the experiment of Fig. 2 indicates, then it must be presumed that some additional low density component or components are associated with the crystal. The experiments reported here do not deal directly with this question. But among the additional components of the scintillon must be included a bioluminescent system which is capable of light emission. From the soluble fraction of a cell extract components of a bioluminescent system have been isolated (3, 11, 12), and it is hypothesized that these are incorporated as a part of the active scintillon. Experiments which support this hypothesis are in progress and will be reported elsewhere.

The functional role of a guanine crystal as a component of the scintillon is not obvious. Crystalline guanine serves as a reflector substance in several biological systems (2, 14, 18), but this seems an unlikely role in a small uni- cellular form, since the occurrence of or need for directional emission is not apparent. We would speculate that instead, it functions as a carrier of some sort, or that it may have a role in exciton migration.

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1 2 0 T H E J O U R N A L O F G E N E R A L P H Y S I O L O G Y • V O L U M E 5 1 • 1968

This point also suffers from the fact that no chevron-shaped crystals have been found in Noctiluca (8) even though scintillon activity has been demon- strated in Noctiluca and shown to exhibit properties similar to that in Gonyaulax. Nonluminescent species have also been reported to possess chevron-shaped crystals (22), but since many chevron-shaped crystals not associated with luminescence occur in Gonyaulax (Fig. 5), it may be that their relationship to bioluminescence is not obligatory.

The hypothesis that the scintillon is responsible for the luminescent flash of the living cell has recently been challenged by Sweeney and Bouck (22) on the basis of measurements of the relative amounts of extractable bioluminescence activity attributable to scintillons compared to the soluble system. They reported a greater yield of luminescence from the soluble system, and on this basis questioned the central role of scintillons in the bioluminescence of dinoflagellates. We were unable to confirm this result, finding instead that the total amount of extractable luminescence activity which both they and we reported constitutes only a small fraction of the activity demonstrable in the living cell (13). As might be expected, the apparent relative proportions of the two activities which one obtains depend on various factors, especially the conditions of assay and the degree of purification.

Even assuming that data of this kind are indicative of the condition in the living cell, it must be recognized that an in vitro measurement of the relative yield of the two systems does not bear critically on the question of function in the intact cell. The matter of primary relevance to be considered is the nature of the control of the reaction: how the bioluminescence is turned on and off with speed and precision to result in the brief brilliant burst of light. A large quantity of free enzyme and substrate could be present in the cytoplasm without being under the control of the flashing mechanism. The organization of the chemical components into a particle is clearly an appropriate method for the compartmentalization and control. The emission in the living cell comes from discrete points, and we have hypothesized that these points correspond to the structures which have been isolated and studied as scintillons.

The actual method for control of the reaction is not understood in any detail. In broad outline it is now known from the work of Eckert on Noctiluca (7) that the luminescent flash is triggered by a bioelectrical event, which may be measured as an action potential of the cell membrane. He speculates that this may act by causing a change in permeability of the membrane which encloses the luminescent organelle, a view which we also have advanced (10, 13). In Gonyaulax the reaction could be triggered by the entry of H + ion into the scintillon. The intensity of the flash would then be governed by the number of H+ ions entering, which would presumably be fewer than the number of luciferin molecules. The decay time of the flash, which is notable for its relative constancy, would be related to the utilization of these protonated luciferin molecules, the kinetics being a defined property of the system.

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RICHARD DESA AND J. WOODLAND HASTINOS Characterization of Sdntillons I2I

A few final comments may be made with regard to the photon yield of the scintillon. This may be calculated from the experiment of Fig. 2 to be about 800. This number represents the average value for the isolated particle, and does not itself give a clue as to whether or not all particles are active. In recent experiments in which scinfillon emission in vitro was observed directly with an image intensifier a photon yield of about 500 quanta per particle was estimated (21).

The question as to whether or not the in vivo flash of the scintillon is of the same intensity or brighter has not been answered. Estimates by Eckert and Reynolds (8) for the in vivo emission from individual points of luminescence in Noctiluca are about two orders of magnitude greater, in the range of 105 quanta per unit, so it may be that the isolated scintillon has lost much of its activity. This loss might involve the complete inactivation of 99% of the particles, leaving behind 1 c-/o in the fully active form. Alternatively, 100% might remain but each with only 1% of its activity. It will probably be possible to distinguish between these alternatives by experiment.

This paper is based on a thesis by Dr. DeSa presented in partial fnifiUment of the requirements for the Ph.D. degree at the University of Illinois. Dr. DeSa held a predoctoral fellowship from the National Institutes of Health. The research was also supported in part by a grant from the National Science Foundation and by a contract from the Office of Naval Research, Nonr 1834 (34).

Received for publication 19 May 1967.

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Bioluminescent particles from the marine dinoflagellate ...€¦ · dinoflagellates (3, 11, 12) and in other systems, notably the firefly (17). Knowl- edge of the physiological aspects