Bioluminescence of Marine Organisms

Chapter 4 Bioluminescence of Marine Organisms TOSHIO GOTO

I. Introduction II. The Luciferin-Luciferase Reaction and Photoprotein III. Cypridina A. The Luciferin-Lucif erase System B. Mechanism of Cypridina Bioluminescence IV. Bioluminescent Coelenterates A. Photoproteins B. The Luciferin-Lucif erase System C. Sensitized Bioluminescence D. Lumisomes: Bioluminescent Particulates V. Bioluminescent Shrimps A. Meganyctiphanes B. Oplophorus and Heterocarpus VI. Bioluminescent Fishes A. Fishes Having Cypridina Luciferin B. Fishes Having Oplophorus Luciferin VII. Bioluminescent Mollusks A. Pholas Dactylus B. Watasenia Scintillans C. Ommastrephes Pteropus VIII. Bioluminescent Worms A. Chaetopterus B. Odontosyllis C. Balanoglossus IX. Luminous Bacteria A. Bioluminescence System B. Bioluminescence Intermediates C. Bacterial Luciferase D. Chemical Reactions for Light Emission E. Formation of Luciferase X. Bioluminescent Dinoflagellates A. The Luciferin-Luciferase System B. Scintillons—Bioluminescent Particles C. Circadian Rhythm of Bioluminescence References

. . . .

" . . .

180 180 181 182 187 193 194 196 198 202 202 202 203 204 204 205 205 205 206 208 208 209 209 209 210 210 211 212 212 214 215 215 215 216 216 179

MARINE NATURAL PRODUCTS Copyright © 1980 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-624003-5

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Toshio Goto

I. INTRODUCTION

It comes as a real surprise that it has been known since ancient times that many luminous organisms produce excited-state molecules and utilize them as a light source. Another amazement is that the efficiency of conversion of chemical energy to light is extremely high; for example, the quantum yield of firefly bioluminescence is 0.88. Such high efficiency has never been obtained by electric lights or by chemiluminescence. In general, bioluminescence is produced by oxidation of a substrate with molecular oxygen similar to the usual chemiluminescence. McElroy and Seliger (1962) suggested that evolution of bioluminescence was initiated in order to remove effectively traces of toxic oxygen when the atmosphere was still in an anaerobic condition. Nowadays many luminous organisms, however, utilize their light for more advanced purposes. Thus fireflies use their light for pheromonal purposes, and deep-sea angler fishes use it to attract prey. A novel explanation of the purpose of pony fish bioluminescence (Hastings, 1971) maintains that the fish emits dim light from all over its ventral surface during daytime to camouflage its silhouette. Although several well-known terrestrial luminous organisms such as the firefly and luminous mushrooms have been described, most bioluminescent animals have been found in the sea; they are distributed over almost all phyla of the animal kingdom from protozoans to vertebrates (Table 1).

II. THE LUCIFERIN-LUCIFERASE REACTIONS AND PHOTOPROTEINS In 1885, Dubois observed in vitro bioluminescence of the luminous beetle Pyrophorus by mixing a hot aqueous extract of the insect with a cold aqueous extract that had been left standing until the initial luminescence ceased. The hot extract contained a heat-stable substrate (general name: luciferin), whereas an enzyme (general name: luciferase) was present in the cold extract; such a reaction has been called the luciferinluciferase (L-L) reaction. Usually oxygen is necessary for bioluminescence. Thus, light is emitted as a result of the enzyme-catalyzed oxidation of luciferin with oxygen. Harvey (1952) had attempted to discover this luciferin-luciferase reaction in many other luminous organisms and found that the following showed positive L-L reaction: the American firefly Photinus, the ostracod crustacean Cypridinay and the Bermuda fireworm Odontosillys. Since then several other organisms have been found to give the L-L reaction (Table 1), but many others have not exhibited such a reaction. Among these, some have recently been shown to have a luminescent protein (general name: photoprotein) that emits light by the action of a low molecular triggering substance.

181

4. Bioluminescence of Marine Organisms TABLE 1 Classification of Marine Bioluminescent Organisms Phylum · Class · Order Bacteriophyta Protozoa Dinoflagellata Cnidaria Hydrozoa Scyphozoa Anthozoa Ctenophora Annelida Polychaeta Mollusca Pelecypoda Gastropoda Cephalopoda Arthropoda Crustacea Ostracoda Euphausiacea Decapoda Hemichordata Vertebrata Osteichthyes (Fish) Myctophina Batracoidida Percina Berycomorphi Stomiatina Ceratiina

Typical organism

Luminous system"

Photobacterium

L

Gonyaulax

L

Aequorea (jellyfish) Pelagia (jellyfish) Renilla (sea pansy) Beröe (jellyfish)

P L P

Chaetopterus (lugworm) Odontosyllis (marine fireworm)

P L

Pholas (boring mollusk) Plocamopherus Watasenia (firefly squid) Loligo (squid)

L

Cypridina (sea firefly) Meganyctiphanes (shrimp) Oplophorus (shrimp) Balanoglossus

L P L L

Neoscopelus Porichthys Apogon Monocentris Polypnus Himantolophius

L L L B B B

P? B

° L: luciferin-luciferase system; P: photoprotein; B: symbiosis with luminous bacteria.

Firefly bioluminescence is one of the systems that has been studied most extensively, though it is not discussed in this chapter (McElroy and DeLuca, 1973). III. CYPRIDINA

There are many bioluminescent organisms that belong to Crustacea. Among those the ostracod Cypridina hilgendorfii Müller has been studied most extensively.

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Toshio Goto

A. The Luciferin-Luciferase System Cypridina hilgendorfii (Fig. 1) is abundant along the coast of Japan. The organism is about 3 mm long and emits a strongly luminescent secretion into the seawater when it is disturbed. It gathers on dead fishes at night and hence can be caught by using dead fishes as bait. The luminescent activity is preserved almost indefinitely in completely dried Cypridina, and the luminescence is restored by moistening (Harvey, 1952). The L-L reaction bf Cypridina was discovered by Harvey in 1917 (Harvey, 1952). Around the coasts of Southeast Asia and the Southern Pacific were found Cypridina noctiluca Kajiyama (Haneda, 1955) and C. serrata Müller (Tsuji et al., 1970), which are smaller than C. hilgendorfii. Vergila harveyi is found around the islands of the West Indies (Kornicker and King, 1965). Luciferin and luciferase of C. noctiluca and C. serrata cross-reacted with those of C. hilgendorfii, hence the luciferins of these organisms are possibly identical, but luciferase of C. serrata could be differentiated from C. hilgendorfii by an immunological method (Tsuji et al., 1970). /. Isolation of Cypridina Luciferin Cypridina luciferin could easily be extracted from dry Cypridina with methanol, but purification was difficult because of its extreme instability.

Fig. 1.

Cypridina

hilgendorfii.

4. Bioluminescence of Marine Organisms

183

Prior to 1950 many research results had been published based on very crude luciferin, which suggested that the substance could be a proteose, a phospholipid, a polyhydroxybenzene, a reduced quinone, or a kind of flavin (Tsuji et al., 1955). The most effective purification of luciferin that had been realized was the Anderson benzoylation cycle (Anderson, 1935); dry Cypridina y after being defatted with benzene, is extracted with methanol to give crude luciferin, which is benzoylated in «-butanol with benzoyl chloride without the addition of base. The benzoate was extracted with benzene and hydrolyzed with hydrochloric acid to recover original luciferin in the aqueous layer. Repetition of this benzoylation cycle afforded "purified" luciferin, 2000 times as active as dry Cypridina. Mason (1952) obtained approximately 20 different amino acids from the hydrolyzate of this "purified" luciferin and suggested that the luciferin is a polypeptide. Shimomura et al. (1957) further purified the "purified" luciferin by cellulose column chromatography; they acidified a methanol solution of the luciferin fraction with concentrated hydrochloric acid, when beautiful orange crystals of Cypridina luciferin dihydrochloride separated, which were 37,000 times as active as dry Cypridina and gave only a few amino acids on acid hydrolysis, although its ultraviolet spectrum was practically identical with that of "purified" luciferin. Thus, there was an amazing difference between "purified" and crystalline luciferin. Later Haneda et al. (1961) developed a new extraction method. After collection, live Cypridina were immediately frozen in dry ice and stored at -20°. Extraction was carried out with methanol containing dry ice. After being defatted with benzene, the crude luciferin was purified by alumina or cellulose column chromatography and then crystallized according to Shimomura's method. The yield was very much improved: about 20 mg of crystalline luciferin dihydrochloride could be obtained from 1 kg of wet Cypridina. 2. Structure of Cypridina Luciferin The structure of Cypridina luciferin was finally elucidated by Kishi et al. (1966a,b) as 1 in the following way. The presence of an indole nucleus was shown by the uv spectrum of hydroluciferin (4), which was obtained from luciferin (1) by catalytic hydrogenation (Shimomura et al., 1957). The amino acid composition of the hydrolysate of 1 and 4 was determined as shown in Table 2 (Eguchi, 1963); this suggested that three independent parts that produce glycine, isoleucine, and arginine, respectively, exist in 1 and 2. When luciferin was oxidized in the presence of luciferase (bioluminescence produced) or bases (nonenzymatic), oxyluciferin (2) and etioluciferin (3) were produced. Acid hydrolysis of 2 gave 3, whose structure was suggested mainly by uv, nmr, and mass spectral analyses

184

Toshio Goto TABLE 2 Amino Acids Obtained from Luciferin (1) and Hydroluciferin (4) Luciferin (1) In vacuum

Glycine Arginine" Isoleucine" Ammonia

0.96 mole 0.62 0.16 0.27

Hydroluciferin (4) In air

In vacuum

In air

0.93 0.38 0.16 0.33

0.03 0.03 0.98 0.40

0.12 0.41 0.96 0.41

a

Combined amounts of arginine, γ-guanidinobutyric acid, and proline. Combined amounts of isoleucine and alloisoleucine; both acids are produced in nearly the same amounts. b

and then proved by synthesis (Kishi et al., 1966c). Based on the structure of 3, the structure of luciferin was suggested to be 1, which was also proved by synthesis (Kishi et al., 1966c). The absolute configuration of the sec-butyl side chain was determined to be the same as that in L-isoleucine by amino acid analysis of the acid hydrolysate of hydroluciferin (4) after treatment with D- or L- amino acid oxidase (Kishi et al., 1966a). Three tautomeric structures, A, B, and C, can be written for the dihydroimidazopyrazinone nucleus involved in C. luciferin. In neutral solution, 2-methyl-3,4-dihydroimidazo[ 1,2-a]pyrazin-3-one exists as tautomer A. In weakly acidic methanol it exists as monocation D,

Luc if erase

II

I]

H

NH2

|Η2/ΡΙΟ2

IJ v H

π

ΙΓΠΓ

V^^NH-ct"H2 NH 2

H Cypridina Oxyluciferin (2)

Cypridina Luciferin (1 )

|[

N^NH H

H 3 cf

N

NH 2

Cypridina Hydroluciferin ( 4 )

Scheme 1

NH-C:

:NH2 -NH2

Cypridina Etioluciferin ( 3 )

185

4. Bioluminescence of Marine Organisms

Ύ

HO

r- C H 3

CH3

0^.

H -CH3

N

B

°w CH3

(Y ^ H

A

(neutral)

Me0H/

^ ™ NnSr*»™

WH3 (γ

H E O2NHC0

^ £ " 2 nr

xMeOH

H in H2O

H in MeOH

cr

HO

CH3

THo

Cf'

H D O0.2N HCl) *ii??H380nm

H r (>12N HCl) λ ^ ) 385nm

r=r

HOv__^H3

cr

v=t 3

Η(λ

"0>=_XH3

^N

G

' H

Scheme 2

whereas in aqueous solution as monocation E. In both strongly acidic aqueous and methanolic solutions it exists as dication F. The NH group in A is acidic (C. luciferin has a pKa at 8.3 in water) and can dissociate to form anion H in basic solutions. Detailed comparison of the uv spectra of C luciferin (1) and of a model compound indicated that the tautomeric forms of C. luciferin are same as those of the model compound (Goto et al., 1975). Curiously, the color of crystalline luciferin dihydrobromide (faintly brown) is quite different from that of the dihydrochloride. This difference is best interpreted by assuming structure D for the dihydrochloride and

Toshio Goto

186

structure G for the dihydrobromide. Solutions of the dihydrobromide have the same orange yellow color as those of the dihydrochloride. 3. Synthesis of Cypridina Luciferin From a biogenetic point of view, C. luciferin (1) consists of three amino acids or their equivalents; namely, tryptophan (or tryptamine), L-isoleucine, and arginine. A synthesis of optically active luciferin was carried out along these lines through etioluciferin (3) as shown in Eq. (1) (Kishi et al., 1966c). In this synthesis, reduction of the pyrazine ring in etioluciferin is necessary prior to condensation with the keto acid; the yield of luciferin was low (ca 1%). Two routes for the synthesis of the 3,7dihydroimidazo[l,2-a]pyrazin-3-one ring system have been developed. One consists of the condensation of a 2-bromopyrazine derivative and an α-amino acid ester followed by acid catalyzed cyclization [Eq. (2)] (McCapra and Chang, 1967) and the other of the condensation of a 2-aminopyrazine derivative, an aldehyde, and sodium cyanide followed by base catalyzed condensation [Eq. (3)] (Goto et al., 1968a). These methods, however, are difficult when applied to the synthesis of luciferin itself. It was later found that the condensation of etioluciferin with the isoleucine moiety could be accomplished by using the keto aldehyde instead of the keto acid [Eq. (4)] (Inoue et al., 1969); as a result, the yield of racemic luciferin was improved up to 70%. McCapra and Roth (1972) synthesized a luciferin analog by a route very similar to the biogenetic pathway from a peptide containing a dehydroamino acid [Eq. (5)]. Hv^0H 2 N N ^NH

1) OH

Η,Λ/^NHCOPh

/ΝγΒΓ

2)

NH2 Rl-^HCOORa

-

C0Ph

1) Al-Hg

+

»NH * < 'NH2 £

2) D C C

-*(Z)-

(1)

HOOC-CH-R! J ^ m

«3

T-iTRi ^ Ν

H+ , R

3 fl

^N^NH 2 if V " RiCHO + NaCN

*f*\

Rä cc-

NC-CH-R! (f**^fslH OH *R2

* R i VA RiCOCHO

/HOOC^

(2)

R

2

//

(3) (4)

(5)

4. Bioluminescence of Marine Organisms

187

4. Cypridina Luciferase Cypridina luciferase has not been obtained in a crystalline state, but in almost pure form (Shimomura et al., 1961, 1969; Tsuji and Sowinski, 1961); it is a simple hydrophobic protein with a molecular weight of about 53,000 (Shimomura et al., 1969) or 68,000 (Tsuji et al., 1974). Characteristics of C. luciferase are isoelectric point 4.35; optimum pH 7.5; Michaelis-Menten constant (25J) 0.52 x 10~6; optimum salt concentration 0.05-0.07 mole/liter NHJ, Na+, or Ca2+; no active SH; and no inhibition with KCN, sodium diethyldithiocarbamate, and 8-hydroxyquinoline. Lynch et al. (1972) reported that Ca2+ is essential for the activity of C. luciferase. B. Mechanism of Cypridina Bioluminescence Contrary to firefly bioluminescence, which requires Photinus luciferin, P. luciferase, molecular oxygen, Mg2+, and ATP and shows product inhibition, the kinetics of Cypridina bioluminescence is very simple, and Harvey observed as early as 1919 that the in vitro luminescence of Cypridina is a first-order reaction without any product inhibition (Harvey, 1952); it involves only Cypridina luciferin, C. luciferase, and molecular oxygen (Johnson et al., 1962). Thus, it is regarded as the simplest bioluminescence system (Chase, 1966). Luciferase

LH + 0 2 (LH: luciferin)

► Product(s) + hv

/. Chemiluminescence Cypridina luciferin gives no light in aqueous solutions without C. luciferase, but it exhibits spontaneous chemiluminescence when dissolved in dimethyl sulfoxide (DMSO) in the presence of oxygen, although the quantum yield is very low (about 0.15% of bioluminescence) (Johnson et al., 1966). When diethylene glycol dimethyl ether (diglyme) containing a trace of acetate buffer (pH 5.6) is used as the solvent, the quantum yield increases to more than 10% ofthat of bioluminescence (Goto, 1968). This high chemiluminescence efficiency coupled with the mild conditions suggests that both chemi- and bioluminescence involve similar mechanisms. McCapra and Chang (1967) studied the chemiluminescence of the 6-phenyl-2,8-dimethyl derivative (5, R = CH3) in DMSO in the presence of potassium ter/-butoxide. They isolated as the reaction product 2-acetamino-3-methyl-5-phenylpyrazine (6, R = CH3), whose fluorescence spectrum in alkaline medium corresponds to the chemiluminescence spec-

188

Toshio Goto

trum, and suggested a mechanism involving a 1,2-dioxetane intermediate. Kinetic measurements supported this mechanism (Goto et al., 1968a). C. luciferin in diglyme containing acetate buffer (pH 5.6) produced chemiluminescence at a rate which was strictly first order with respect to luciferin and to oxygen. The luminescence spectrum corresponded to the fluorescence spectrum of neutral oxyluciferin (2) (Goto et al., 1968b). Spectral investigation indicated that the excited-state product which is first formed is the anion of oxyluciferin, which is then protonated to form neutral oxyluciferin in the excited state.

(5)

R=H R=CH3

(6)

R=H R=CH3

In summary, C. luciferin (1) dissociates to its anion (pKa 8.3 in water), which is then oxidized by molecular oxygen to give a hydroperoxide anion. The anion decomposes through a dioxetane intermediate to form oxyluciferin (2) anion in a singlet excited state. In neutral solutions protonation occurs on the excited anion to give the excited neutral oxyluciferin molecule (2), which subsequently emits light (scheme 3). The oxidation of the luciferin anion with molecular oxygen in aprotic solvents proceeds in a radical nonchain mechanism; luciferin anion gives its electron to molecular oxygen to form, in a cage, a pair of luciferyl and Superoxide anion-radicals, (rate-determining step), which combine rapidly to form hydroperoxide anion (Goto, 1968). LH <==t

L:- — ^ - »

L·

02-

> L—OO"

C. luciferin is autoxidized in aqueous solutions without enzyme to form red-colored "reversibly oxidized luciferin" (Harvey, 1952) (luciferin-R; Goto, 1968), which can be formed also by oxidation with ferricyanide, lead dioxide, or diphenyl picryl hydrazyl radical (DPPH), and reduced to C. luciferin (1) with sodium hydrosulfite or sodium borohydride (Shimomura et al., 1957). It is further oxidized slowly to colorless compounds of unknown structure, but in the presence of luciferase it emits light, although the rate is very much slower than that of luciferin (Goto et al., 1973a); it had long been thought to have no luminescent activity. Since only one mole of DPPH radical was necessary to produce luciferin-R, which gave no esr signal, it was suggested to be a dimer of the luciferyl

189

4. Bioluminescence of Marine Organisms Ri

χτ ^ xr

*2

(1)

o—o

0—0

o
-R,

02

or sJ

0

XX

R3

l

(A)

^

Rl

-N- 'R 2 (B)

J

0 -Ri

0^

XT"

XT

+ co2

R3

°γ-«ι

ίΤ

hi

R3^N^R2 (2) Scheme 3

radical (Goto, 1968). Luciferin-R is also formed during chemiluminescence even in aprotic solvents when the concentration of luciferin is high (Goto et al., 1973a). LH-

-» L·

L—L

Two structures are possible for luciferin hydroperoxide, that is, A and B. Structure A is now widely accepted, but structure B might be preferred, since the luciferin chemiluminesces under very mild conditions; in the case of the 2-methyl-6-phenyl analog (5, R = H) it occurs even in aqueous solutions at pH 5.6 (acetate buffer) in the presence of hydrogen peroxide and ferric ions. Incidentally, the hydroperoxides of lophine and skatole require strongly basic conditions for light production, whereas some oxalic esters chemiluminesce without addition of base, when they react with hydrogen peroxide to form their half-peracids (Rauhut, 1969). This difference may be explained in terms of the acidity of the hy-

190

Toshio Goto

droperoxides (pKa in the range of 11-12) and the peracids (pKa between 7 and 8): dissociation is necessary for luminescence. 2. Bioluminescence The Cypridina L-L reaction (Fig. 2) is strictly first-order with respect to luciferin, and the rate is proportional to the quantity of luciferase. One molecule of luciferin reacts with one molecule of oxygen to produce one molecule each of oxyluciferin and C0 2 (Stone, 1968); the quantum yield of luminescence is 0.28 ± 0.04 (Johnson et al., 1962; Shimomura and Johnson, 1970). Shimomura and Johnson (1971) proved the involvement of a dioxetane intermediate; when C. luciferin was oxidized with 18 0 2 in the presence of luciferase, 0.8 atom of 18 0 was incorporated into the C0 2 produced. If open-chain hydroperoxide were involved instead of the dioxetane, no 18 0 would have been incorporated. Although C. oxyluciferin (2) gives strong fluorescence in aprotic solvents, almost no fluorescence is observed in aqueous solutions. This is one of the reasons that luciferin 1 does not give light in aqueous solutions without the enzyme. The presence of an enzyme-oxyluciferin complex was assumed (Goto et al., 1968b), in which the emitter, excited-state oxyluciferin, is in an environment similar to that in aprotic solvents (hydrophobic environment). Indeed, addition of purified enzyme to an

Fig. 2. Cypridina L-L reaction.

4. Bioluminescence of Marine Organisms

191

aqueous solution of 2 enhanced the fluorescence intensity of 2; a 1:1 complex between 2 and luciferase was formed (Shimomura et al., 1969). An oxyluciferin analog, 2-acetamido-5-phenylpyrazine (6, R = H), was found to be strongly fluorescent in aqueous solutions as well as in aprotic solvents, and hence the corresponding luciferin analog, 2-methyl-5-phenyl derivative (5, R = H), should give light in aqueous solutions if its hydroperoxide is formed. Indeed, it gave light in aqueous solutions in the presence of hydrogen peroxide and ferric or ferricyanide ions, but not with molecular oxygen (Goto, 1968). Accordingly, the enzyme activity consists not only of the enhancement of fluorescence intensity of oxyluciferin, but also of the enhancement of the reaction rate of luciferin with molecular oxygen, similar to the action of aprotic polar solvents. Thus, C. lucif erase is regarded as a hydrophobic enzyme, as arePhotinus luciferase (hydrophobicity 1240 cal/residue; Denburg and McElroy, 1970) and Renilla luciferase (1200 cal/residue; Matthews et al., 1977). Consideration of these factors led to the expectation that if micelles of a suitable surfactant are present, luciferin might be absorbed into the micelle interior formed by the hydrophobic portion of the surfactant, and produce light in aqueous solutions, as it does in the presence of the enzyme (biomimetic luminescence). Indeed, chemiluminescence of luciferin was observed in aqueous micelle solution of cetyl-trimethylammonium bromide (cationic), but not in the micelle solution of lauryl sulfate (anionic), although oxyluciferin showed strong fluorescence in both micelle solutions. The reason could be that the anionic surfactant forms micelles with highly charged negative surfaces, which prevent luciferin anion from entering the micelle interior, or in which anionradical-type oxidation reaction does not proceed (Goto and Fukatsu, 1969). The specificity of the enzyme was tested by using modified luciferins as the substrates (Table 3) (Goto et al, 1973b). Change in the length of the chain between the guanidine and the imidazopyrazine ring produced a dramatic change in the rate of bioluminescence, but it had no effect on the chemiluminescence rates. Luciferin analogs with an indole group gave bioluminescence efficiency about 10 times better than chemiluminescence efficiency, but no such enhancement was observed in the analogs with a phenyl group; the indolyl group is thus essential for the high quantum yields of bioluminescence. 3. Dioxetane Intermediate Since quantum yields of bioluminescence are usually very high (Table 4), decomposition of 1,2-dioxetane intermediates involved in bioluminescence must produce the emitters in a singlet excited state. Simple 1,2-

192

Toshio Goto TABLE 3

Effect of the Substituents on the Imidazopyrazine Ring on the Rate and Light Yield of Bioluminescence and Chemiluminescence

τ^T ί"Ύ A il

~ ~ ^ ^

.N

H

-R2

Relative rate of luminescence

Ri

3-Indolyl 3-Indolyl 3-Indolyl 3-Indolyl 3-Indolyl 3-Indolyl Phenyl -H 3-Indolyl Phenyl a b

R2

Bioluminescence

-(CH2)2-G« -(CH2)3-G -(CH2)4-G -(CH2)5-G -(CH2)7-G -(CH2)3-NH2 -(CH2)3-G -(CH2)3-G -H -H

1 1006 1 4 0.6 0.2 0.4 0 0.1 0

Chemiluminescence 100 100* 100 100 100 100 200 0.01 30 (25)

Ratio of »

l i n n t \/ΙΑ1/Ί

ugm yiciu Bioluminescence/ Chemiluminescence 10 10 10 10 10 1 0.5

G = guanidino group. Taken as the standard.

dioxetanes such as trimethyl-l,2-dioxetane are fairly stable at room temperature and yield on thermal decomposition two molecules of carbonyl compounds, one of which is mainly in the triplet excited state (review, Hastings and Wilson, 1976). Adam and Lin (1972) synthesized 4-tertbutyl-l,2-dioxetan-3-one in the expectation that it might give a high yield of singlet excited-state molecules, since such a dioxetanone structure has TABLE 4 Quantum Yields of Bioluminescence Source Bacteria Cypridina Renilla Firefly Aequorea

Yield 0.12-0.17 0.28 0.05 0.88 0.23

193

4. Bioluminescence of Marine Organisms \J

\J

|-Ri

0J

R

3

^N^

?co2 · γ ι

R 2

7^

co2

°^Ri

IT

Scheme 4

been assumed to be involved in the intermediates of firefly, Cypridina, and coelenterate bioluminescence. This compound, however, on thermal decomposition produced mainly triplet state molecules. Recently, McCapra (1977) and Schuster (Koo et al., 1978) proposed a mechanism in which an electron-donating group on the dioxetane plays an important role in the production of high quantum yields of singlet excited-state molecules. According to this mechanism decomposition of the dioxetanone derived from C. luciferin is illustrated in Scheme 4. IV. BIOLUMINESCENT COELENTERATES Coelenterata (phylum: Cnidaria) are divided into three classes: Hydrozoa, Anthozoa, and Scyphozoa; all of them include bioluminescent organisms. The hydrozoan Aequorea aequorea (jellyfish) has a photoprotein, aequorin, which emits light by the action of Ca2+ without molecular oxygen. On the other hand, the anthozoan Renilla reniformis (sea pansy) has a luciferin-luciferase system and requires molecular oxygen for light production. It contains, as the precursor of luciferin, Renilla luciferyl sulfate, which is hydrolyzed by luciferin sulfokinase to free Renilla luciferin. The luciferin emits light in the presence of Renilla luciferase and molecular oxygen. Renilla also has a luciferin-binding protein (LBP) and a green fluorescent protein (GFP). The former protein acts as storage for luciferin; free luciferin combines with this protein to give an inactive form, which supplies luciferin in the presence of Ca2+. Thus, Renilla luminescence is also triggered by Ca2+. Aequorea photoprotein and the Renilla luciferin-luciferase system give blue light centered around 470490 nm, whereas in vivo bioluminescence of these organisms is green (\max 509 nm). An energy transfer from the emitter to the green fluorescent protein was suggested; Aequorea also has the GFP. The Renilla bioluminescence system is included in a particulate called lumisome. A similar system has been found in other anthozoa: Renilla mülleri, R. kollikeri, Cavernularia obesa, Ptilosarcus guerneyi, Stylatula elongata, and Acanthoptilum gracile (Cormier et al., 1973). Aequorinlike photopro-

194

Toshio Goto

teins, halistaurin and obelin, were isolated from Halistaura (Shimomura et al., 1963c) and Obelia geniculata (Campbell, 1974; Moisescu et al., 1975), respectively. In the phylum Ctenophora, Ca2+-triggered photoproteins similar to aequorin have been isolated: two soluble photoproteins, mnemiopsin I (MW 24,000) and II (MW 27,500), from jellyfish of Mnemiopsis sp. (Ward and Seliger, 1974a,b, 1976), and one, berovin (MW 25,000), from Bero'e ovata (Ward and Seliger, 1974a,b). These photoproteins give the same in vitro emission at Xmax 485 nm, whereas in vivo emissions of Mnemiopsis and Bero'e are at Xmax 488 nm and 494 nm, respectively; these organisms have no green fluorescent protein. Girsch and Hastings (1978) also reported isolation of mnemiopsin (MW 23,000) from Mnemiopsis leidyi. These photoproteins are light inactivated (Ward and Seliger, 1974b). A. Photoproteins Bioluminescence of the jellyfish Aequorea aequorea has been known for a long time, but attempted isolation of the substances showing L-L reaction had failed. In 1962 Shimomura ef al. (1962, 1963a,b) succeeded in isolating a kind of luminescent protein. Thus, jellyfish photophores were extracted with EDTA solution, and the protein (general name: photoprotein) was precipitated from the extract by addition of ammonium sulfate and purified by DEAE-cellulose chromatography; it was named aequorin (MW about 31,000; Kohama et al, 1971). The photoprotein emits light by addition of a trace of Ca2+ or Sr2+ without interference from Mg2+. This specific character has been applied to the detection and microdetermination of Ca2+ and Sr2+ (limit of detection: 10~8 gm Ca2+/ml solution or about 10"7 M Ca2+) (Shimomura et al., 1963d). Molecular oxygen is not necessary in this light production; the same luminescence can be observed even under a hydrogen atmosphere. Aequorin contains enough energy to produce light but needs a trigger substance such as Ca2+ to initiate light production. Hastings (1968) classified the photoprotein as a "precharged system." For light production the photoprotein must have some chromophore in its molecule to produce a light-emitter with fluorescence ability, but such a chromophore could not be isolated directly from aequorin. Denaturation of aequorin separated a low-molecular-weight fluorescent compound, coelenteramine (9) (Shimomura and Johnson, 1972). Aequorin itself is nonfluorescent, but by Ca2+-triggered luminescence afforded a blue fluorescent protein (BFP), which could be separated by acid, urea, ether, butanol, or gel filtration into two components, coelenteramide (8) and an

4. Bioluminescence of Marine Organisms

195

apoprotein (Shimomura and Johnson, 1973). The BFP could be regenerated by mixing coelenteramide and apoprotein in the presence of Ca2+. The structures of coelenteramine (9) (Shimomura and Johnson, 1972) and coelenteramide (8) (Shimomura and Johnson, 1973) were determined chemically and confirmed by synthesis (Kishi et al., 1972; Shimomura and Johnson, 1973). Surprisingly these compounds are structurally closely related to Cypridina etioluciferin (3) and oxyluciferin (2), respectively. This similarity between the two series of compounds led to the expectation that the chromophore in aequorin would be 7 or its derivative; Shimomura et al. (1974a) named the postulated compound coelenterazine (7). Indeed, aequorin could be regenerated by incubation of the apoprotein with coelenterazine (7) synthesized by Inoue et al. (1975b) in the presence of oxygen (Shimomura and Johnson, 1975). The yield of aequorin was approximately 50% after 30 minutes. Although in the presence of Ca2+ the system containing BFP, 7, and molecular oxygen gave luminescence similar to the L-L reaction (apoprotein may be regarded as luciferase), the system could not produce the bright flashes of light that are observed in live Aequorea, since the rate of regeneration of the photoprotein is very much slower (turnover number : ca 1-2 per hour) than Ca2+-triggered luminescence. Aequorin has a uv-vis absorption spectrum different from that of coelenterazine (Shimomura and Johnson, 1969a) and does not need molecular oxygen to produce light in the presence of Ca2+; the chromophore in the photoprotein must be present in the form of a peroxide that is converted to a dioxetane by the Ca2+ trigger and then emits light. When treated with aq NaHS0 3 , aequorin gave a very unstable yellow compound (YC), whose structure was elucidated to be 10 from the transformations shown in Scheme 5 (Shimomura et al., 1974b; Shimomura and Johnson, 1978). It is easily dehydrated to 13, which was already isolated from livers of Watasenia scintillans (squid) and named Watasenia dehydropreluciferin (Inoue et al., 1977c). Treatment of aequorin with Na2S204 yielded coelenterazine (7). These results suggested a peroxide form of coelenterazine in aequorin. Aequorin contains two thiol groups, at least one of which is necessary for stabilization of the peroxide moiety. The structure of the functional part of aequorin and its luminescence reaction suggested by Shimomura and Johnson (1978) are shown in Fig. 3. Contrary to Shimomura's results, Ward and Cormier (1975) claimed that they extracted Renilla luciferin from aequorin, mnemiopsin, and berovin by treatment with mercaptoethanol, and Hori et al. (1975) suggested that in these photoproteins the luciferin is noncovalently bound to the protein, which contains an oxygenated species, possibly a hy-

196

Toshio Goto

Coelenteramide ( 8 )

Coelenteramine (9) Scheme 5

droperoxide group. Shimomura and Johnson (1978) found that YC can be reduced to coelenterazine by 2-mercaptoethanol treatment and hence the luciferin obtained by Ward and Cormier was possibly an artifact. B. The Luciferin- Luciferase System Cormier (1959) found the L-L reaction in Renilla reniformis, and isolated Renilla luciferyl sulfate (Hori and Cormier, 1965; Cormier et al., 1966b). The sulfate is stable in neutral or basic solutions, but acidification converts it to Renilla luciferin, which then emits light in the presence of

197

4. Bioluminescence of Marine Organisms BFP

AEOUORIN Protein

Oz^ N/ \

\i 1

x

>J

Protein

SH o-q

C0 2

SH

+ LIGHT

2Ca 2+

^ ^

°vrY^ii W^OH

/NUN Π i

^NHXX0H

7 fr*NiS

HO'

kjy^ A

ps

2Ca',2+

^

Coelenteramide 02

H

1

Coelenterazine

HO'

Fig. 3. Postulated mechanism of luminescence and regulation of aequeorin (Shimomura and Johnson, 1978).

Renilla luciferase to afford Renilla oxyluciferin as the final product. The luciferin can also be obtained by anaerobic incubation of the sulfate with crude enzyme preparation, Ca2+, and 3\5'-ADP; the acidic sulfate group is removed in this reaction. Thus bioluminescence proceeds in two steps: in the first dark reaction the sulfate group in the luciferyl sulfate (L—S03H) is transferred to 3',5'-ADP to form free luciferin (LH), which is then oxidized by molecular oxygen to emit light. Enzymes catalyzing these two reactions were isolated from the crude enzyme preparation. One is Renilla luciferin sulfokinase, which catalyzes the first reaction; the other is the luciferase, which catalyzes the second step, as shown in the following equations (Cormier et al., 1966, 1970). L-sulfokinase/Ca2+

L—SO3H + 3',5'-ADP<^LH + Os

R. luciferase

τ

„

Λ A

„0

=± LH + PAPS

»Product + hv

(PAPS = 3'-phosphoadenosine-5'-phosphosulfonic acid)

Renilla reniformis luciferase has been purified (Karkhanis and Cormier, 1971); it has the following characteristics (Matthews et al., 1977): MW 35,000; specific activity, 1.8 x 1015 hv seeding -1 ; turnover number, 111 min -1 ; average hydrophobicity, 1200 cal/residue; optimum tempera-

198

Toshio Goto

ture, 32°; optimum pH, 7.4; about 50% inhibition 10"5 M of Zn2+ and Cu2+; reversible inhibition with Na2EDTA; competitive inhibitor /7-benzyloxyaniline Ki 7.7 μ,Μ; and oxyluciferin Ki 23 nM. Since p-benzyloxyaniline is a competitive inhibitor, Matthews et al. (1977) used p-benzyloxyaniline-Sepharose affinity column chromatography for purification of luciferase. The luciferase is a rather stable, highly hydrophobic enzyme that tends to self-associate to form a high-molecular-weight inactive species, from which attempted regeneration of active lucif erase monomers have failed. The structure of Renilla luciferin had long been questioned (Hori et al., 1972; Hori and Cormier, 1973), but it was recently identified as the same compound as coelenterazine (7), Oplophorus luciferin, and Watasenia preluciferin (Inoue et al., 1977a; Hori et al., 1977). Synthetic Renilla dehydroxyluciferin was found to be fully active (Hori et al., 1973, 1975). The chemical mechanism of Renilla bioluminescence which has been suggested (Matthews et al., 1977) is similar to that of Cypridina (Scheme 6). Interestingly, the light-emitter is oxyluciferin monoanion with an undissociated phenyl group (Hori et al., 1973). In addition to luciferyl sulfate and luciferase, Anderson et al. (1974) isolated from Renilla a protein (MW 18,500) containing one molecule of Renilla luciferin (coelenterazine) noncovalently bound (Cormier and Charbonneau, 1977). Originally the protein was thought to be a photoprotein similar to aequorin (Cormier et al., 1973), but in this case molecular oxygen and luciferase are necessary in addition to Ca2+ to produce light. Now it was found that in the presence of Ca2+, this protein releases its bound luciferin and transfers it to luciferase to give the L-L reaction in the presence of oxygen. Thus the protein, calcium-triggered luciferinbinding protein (LBP) differs from a photoprotein such as aequorin. The uv-vis absorption spectrum of LBP is similar to that of coelenterazine (7), whereas the aequorin spectrum is completely different. Coelenterazine (7) has also been isolated and identified from other coelenterates, Cavernularia obesa (sea cactus) and Ptilosarcus guerneyi (sea pen) (Inoue et al., 1979a). C. Sensitized Bioluminescence In vitro bioluminescence of the photoprotein aequorin and Renilla luciferin emits blue light, which has a broad maximum centered around 470-490 nm, but Aequorea and Renilla show green/« vivo luminescence, which appears as a sharp, structured emission peaking at 508 nm (Figs. 4 and 5). These coelenterates have a green fluorescent protein (GFP) (MW

199

4. Bioluminescence of Marine Organisms

R. dehydroxyluciferin ( synthetic)

Renilla luciferyl sulfate L. Sulfokinase 3·.5·-ΑΟΡ Ca 2+

Nerves rote in

GFP

* ■> GFP - > hv

R. oxyluciferin monoanion ( Excited state ) Scheme 6

approximately 40,000), whose fluorescence spectrum matches the green luminescence. Thus sensitized luminescence involving energy transfer from excited oxyluciferin to GFP is suggested in these cases (Wampler et al., 1971, 1973; Cormier et al., 1973; Morise et al., 1974). Addition of GFP to aequorin or to the Renilla luciferin-luciferase system yielded green bioluminescence. In the Renilla case the luminescence quantum yield increases 5.7-fold by this sensitization (5.7% without sensitization and 30% with sensitization) (Fig. 6). Most bioluminescent coelenterates contain GFP artd produce green light, except Pelagia and Mnemiopsis leidyi, which have no GFP and therefore produce blue emission.

200

Toshio Goto

Fig. 4. Aequorea

aequorea.

201

4. Bioluminescence of Marine Organisms

Fig. 5. Aequorea bioluminescence. 1

~!

1

1

1

1

1

1

"Ί

1

11

1

1.0

0.8

z

-

-

£0.6 o

d

Z

.

v

0.A

LU

> < _l

-

Lü

er 0.2

i~-

A20

A60

j\

500 540 WAVELENGTH ( nm )

.

4 580

620

Fig. 6. The Renilla green emission (solid line) and blue emission (dash line) spectra (Ward and Cormier, 1976).

202

Toshio Goto

Although efficiency of the energy transfer between two molecules is inversely proportional to the sixth power of the distance between them, 2.7 x 10"6 M GFP is enough to produce green light (energy transfer essentially 100%), thereby suggesting association between luciferase and GFP (Morin and Hastings, 1971; Wampler et aL, 1971; Ward and Cormier, 1976). D. Lumisomes: Bioluminescent Particulates Anderson and Cormier (1973) isolated from bioluminescent coelenterates, such as Obelia, Clytia, Renilla, Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus membrane enclosed vesicles (lumisomes) responsible for bioluminescence; the vesicle has a diameter of approximately 0.2 μπι and contains all proteins necessary for green bioluminescence; that is, the calcium-triggered luciferin binding protein, luciferase, and the green fluorescent protein, which is associated with the membrane. The light production is initiated by permeation of Ca2+ into the lumisomal membrane, and the release of Ca2+ is controlled by a nerve net. Permeability of Ca2+ is enhanced by higher concentration of Na+ inside of the membrane. The Na+ gradient dependent Ca2+ transport occurs on a millisecond time scale, which is in accord with a flash light production of Renilla and others. Electron microscopic studies on Renilla miilleri indicated that lumisomes, membrane-enclosed vesicles (0.1-0.2 /z,m), are contained in a large (4-6 μπι) membrane-bounded subcellular organelle, which was named luminelle (Spurlock and Cormier, 1975). Henry (1975) isolated a particulate system similar to Renilla lumisomes from the bioluminescent anthozoan coelenterate Veretillum cynomurum. Morin and Hastings (1971) observed also a similar system in the extracts of the hydrozoan Obelia. V. BIOLUMINESCENT SHRIMPS

Crustacea include Ostracoda (e.g., Cypridina), Euphausiacea (e.g., Meganyctiphanes norvegica), and Decapoda (e.g., Sergia lucens, Oplop hor us, etc.). A. Meganyctiphanes Shimomura and Johnson (1967, 1968a) isolated in fairly pure form a photoprotein (P) and a low-molecular-weight fluorescent compound (F)

4. Bioluminescence of Marine Organisms

203

from euphausid shrimp, Meganyctiphanes norvegica, found along the coast of Scandinavia. When solutions of the two compounds, P and F, were mixed in the presence of molecular oxygen, blue bioluminescence was observed, whose spectrum (Xmax 476 nm) is identical with the fluorescence spectrum of F. The luminescence is sensitive to pH change; luminescence intensity increases from 4% of the maximum value to 95% by changing the pH from 7.4 to 7.6 (phosphate buffer). F is unstable in acidic solution and easily oxidized with oxygen. P is separated by gel filtration into two components, one of which has a molecular weight of approximately 900,000 and the other of which has one of 360,000; both components show the same luminescent activity. The former may be an aggregate of the latter. They are unstable to heat; in a dilute solution, similar to the bioluminescent condition, the activity falls to half for 10-25 min at pH 7.5 and 0°. P is oxidized by molecular oxygen by catalysis of F and oxidized P transfers its energy to F, which then emits light. Thus P is a photoprotein that is oxidatively decomposed to give an excited-state molecule and F is a catalyst without decomposition contrary to the L-L reaction; quantum yields of P are 0.55 (high-molecular-weight component) and 0.22 (low-molecular-weight component), and of F they are more than 10, indicating recycling use of F during luminescence.

B. Oplophorus and Heterocarpus Sergia lucens, pelagic shrimp, abundantly found in Suruga Bay, Japan, has been shown to have photophores, but bioluminescence has never been observed on it (Omori, 1974). Haneda (1955) found an L-L reaction on Oplophorus gracilorostris, a deep-sea decapod rarely found among Sergia lucens, from which crude luciferin and luciferase were extracted (Johnson et al., 1966). Inoue et al (1976a) isolated pure Oplophorus luciferin from each of the decapod shrimps, Oplophorus spinosus (15 /xg from 60 individuals) and Heterocarpus laevigatus (25 /xg from 9 individuals), and identified it as coelenterazine (7). Shimomura et al. (1978) purified luciferase of Oplophorus gracilorostris and examined the L-L reaction. The molecular weight of luciferase is approximately 130,000, which consists of 4 monomers, 31,000 daltons each. L-L reaction with O. luciferin gave light at Xmax 462 nm with production of C0 2 and oxyluciferin (8) (coelenteramide), possibly by the dioxetane mechanism. Oplophorus luciferase is unusually stable to heat, as shown in Fig. 7. Quantum yield of O. luciferin is 0.34 at 22°.

Toshio Goto

204

12 h ^BACTERIA 10h 0PL0PH0RUS ^ 8

>Σ Z>

O

LATIÄ

LU DC

CHAETOPTERUS 20

_L

40 60 TEMPERATURE (°C )

80

Fig. 7. Effect of temperature on the relative quantum yield of various bioluminescent systems (Shimomura et al., 1978).

VI. BIOLUMINESCENT FISHES Bioluminescent fishes are divided into two groups. One involves those luminescing by symbiosis with luminous bacteria, and the other those luminescing by the luciferin-luciferase reaction with luminous substances. Many kinds of fishes of the former type have been found, mostly in Japan (Haneda, 1977)—for example, the order Berycomorphi, which includes Monocentris japonicus, Anomalops katoptron, and Paratrachichthys prosthemius, and the order Percida, which includes Leiognathus equlus, Acropoma japonicum, and Siphamia versicolor. Most of the Siphamia family luminesce with luminous bacteria except Apogon elioti, which uses Cypridina luciferin for light production. A. Fishes Having Cypridina Luciferin Haneda and Johnson (1958) found that Parapriacanthus beryciformes (Pempheridae) and Apogon ellioti (Apogonidae) have luciferin and luciferase. The luciferin was shown to be identical with Cypridina luciferin (Johnson et al., 1961; Haneda et al., 1966), but the luciferase was found immunologically to be different from that of Cypridina (Tsuji and Haneda, 1966). Since the stomachs of the fishes contain Cypridina and

4. Bioluminescence of Marine Organisms

205

since the luminous ducts are connected to the digestive tract, the luciferin in the fishes is possibly of Cypridina origin. Porichthys notatus (Batrachoididae), which is found along the coast of California and called midshipman, has an L-L system (Xmax 485 and 507 nm) which was cross-reacted with those of Cypridina, but in this case no evidence has been obtained for the origin of the lucif erin (Cormier et al., 1967; Tsuji et al., 1971). Porichthys from Puget Sound (Washington state) is nonluminous but becomes luminous by injection or feeding with Cypridina luciferin (Tsuji et al., 1972, 1975; Barnes et al., 1973). B. Fishes Having Oplophorus Luciferin Inoue et al. (1977b) isolated Oplophorus luciferin (7) from livers of Myctophinafish,Neoscopelus microchir (2.1 mg from 25 specimens). The luciferase could not be extracted from TV. microchir, but a closely related Myctophina fish, Diaphus elucens, gave the luciferase by extracting with phosphate buffer (pH 7.0). Although the latter fish has almost no free luciferin in its body, a pair of its large nasal photophores yielded O. luciferin (5 ^g/liter specimen) (Inoue et al., 1979b). These results indicate that Oplophorus luciferin is possibly used for light emission of these Myctophina fishes. It is not known whether the luminescent substance comes from the diet or is synthesized by the fish.

VII. BIOLUMINESCENT MOLLUSKS The phylum Mollusca includes Polecypoda (e.g., Pholas dactylus), Gastropoda (e.g., Planaxis, Latia), and Cephalopoda (e.g., Watasenia scintillans). A. Pholas dactylus Pholas dactylus, the boring mollusk, has an L-L system, which was first demonstrated by Dubois in 1887 (Harvey, 1952). Pholas luciferin (MW 34,000) is a glycoprotein having a prosthetic group (Xmax 307 nm; € 11,800) (Henry and Monny, 1977) and emits light with oxygen in the presence of Pholas luciferase or with many reagents that produce Superoxide ion (Henry and Michelson, 1970, 1973; Henry et al., 1973; Michelson, 1973). Pholas luciferase is a metalloglycoprotein (dimeric form: MW 310,000) containing two atoms of copper(II) and has two independent binding sites, which are probably related to its dimeric nature (Henry and Monny, 1977). It has a peroxidase activity capable of oxidiz-

206

Toshio Goto

ing ascorbic acid in the presence of H 2 0 2 (Henry et al., 1973), but in the case of bioluminescence this enzyme acts as an oxidase, which requires molecular oxygen (Henry et al., 1973, 1975). During the bioluminescence the uv absorption band of luciferin disappears, but this change occurs after emission of light and needs ascorbic acid or other reductants (Henry and Monny, 1977). This L-L system is unique, since protein-protein interactions play an important role in the light production (Henry and Monny, 1977). A suggested mechanism for Pholas bioluminescence (Henry et al., 1975) involves reduction of the Cu2+-enzyme with luciferin to form luciferin radical and Cu+-enzyme, which reacts with molecular oxygen to regenerate Cu2+ enzyme and Superoxide ion. Interaction of the luciferin radical and Superoxide ion then gives rise to a luciferin peroxide intermediate, decomposition of which results in light emission. B. Watasenia scintillans Squids (Decapoda) can be classified into Oegopsida and Myopsida; the former squids are found mainly in the deep ocean and the latter in shallow water. Myopsid squids such as Loligo edulis, Euprymna morsei, and Sepiola biostorata utilize the light of luminous bacteria that are cultivated in their photophores (symbiosis) (Harvey, 1952), whereas many oegopsid squids produce intracellular luminescence from their own luminescent systems. One of the most famous luminescent squids is Watasenia scintillans (Fig. 8) (Japanese name: hotaru-ika, meaning firefly squid), which belongs to Oegopsida and is found abundantly in Toyama Bay, on the coast of the Sea of Japan. This squid has three black spots (photophores) on the tips of each of the ventral pair of arms as well as many small light organs scattered over the body. Shima (1927) reported that luminous bacteria could be isolated from the squid, but Kishitani (1928) and Okada et al. (1933) disputed this. This squid is rather fragile and dead squid produce no light; attempted extraction of luminescent systems such as an L-L system or a photoprotein failed. Goto et al. (1974) extracted fluorescent compounds from the arm photophores on the expectation that fluorescent substances might relate to the product of luminescence in analogy to the bioluminescence mechanisms of other L-L reactions and that the products of luminescence might be accumulated in the photophores of dead squid. A fluorescent compound extracted from the arm photophores (4 mg from 10,000 individuals) was shown to have structure 12, which is the disulfate of coelenteramide (8), and is a possible light-emitter of the squid. It was named Watasenia oxyluciferin, although a complete system of

4. Bioluminescence of Marine Organisms

207

Fig. 8. Watasenia scintillans.

bioluminescence has not been extracted in vitro. Structural similarity of Watasenia oxyluciferin and Cypridina oxyluciferin, coupled with consideration of the mechanisms proposed for Cypridina bioluminescence (Goto and Kishi, 1968), suggests that the luminescent moiety supposed to be present in the squid could have the structure 11 or a derivative, which should produce chemiluminescence in aprotic polar solvents (Goto et al., 1968a). After extensive search for such a chemiluminescent substance in lyophylized organs of the squid, it was found that livers of the squid contain fairly large amounts of a strongly chemiluminescent compound, whose structure was determined as 7 by total synthesis (Inoue et al., 1975); this compound has the same structure as that of the assumed chromophore in aequorin, the jellyfish photoprotein (Shimomura et al., 1974a). Its role in Watasenia bioluminescence would be as a precursor of luciferin (11), and hence it is named Watasenia preluciferin. Preluciferin is easily oxidized to Watasenia dehydropreluciferin (13), which was also found in Watasenia livers (Inoue et al., 1977c). Finally, Watasenia luciferin (11) was isolated from the arm photophores by treatment with sodium methoxide in the absence of oxygen (40 μ-g from about 2500 individuals) (Inoue et al., 1976b). These results suggest that luciferin (11) is attached to insoluble material in the photophores and stored in bound form. Light is

208

xna

Toshio Goto

Blood Vessel? HO3SO' Watasenia Preluciferin ( in Liver ) ( 7 )

hy -<-

#

Watasenia Luciferin (11) ( in Luminous Organ ) IO2

Green Fluorescent Compound(G)

»SO3H

HO3SO Watasenia Oxyluciferin ( 1 2 ) ( Excited State )

Scheme 7

emitted from the excited oxyluciferin produced by oxidation of luciferin in the bound state or after liberation (Scheme 7). Oxyluciferin shows fluorescence in water at \ m a x 400 nm; the energy may be transferred to a green fluorescent compound, which exists in the photophores (Goto et al., 1974). C. Ommastrephes Pteropus The pelagic squid Ommastrephes pteropus has a large oval light organ, which gives light at Xmax 474 nm in the presence of molecular oxygen. The bioluminescence system could not be.extracted in vitro, but two highly fluorescent compounds were isolated, one of which gives a fluorescence spectrum matched with that of the bioluminescence (Girsch et al., 1976).

V m . BIOLUMINESCENT WORMS

Many luminous worms have been found on seashores. Chaetopterus and Odontosyllis belong to Annelida; the former has a photoprotein, whereas the L-L system was found in the latter. Balanoglossus, which belongs to Hemichordata, has a peroxidase system for light emission.

4. Bioluminescence of Marine Organisms

209

A. Chaetopterus The marine polychaete annelid Chaetopterus variopedatus lives in a tube in the sand near the seashore. By mechanical or electrical stimulation this worm gives off blue light (465 nm; Nicol, 1957) from luminous glands at the 12th segment and it also has a luminous secretion. The L-L reaction has not been observed with this worm, but Shimomura and Johnson (1966, 1968b) isolated a photoprotein, Chaetopterus photoprotein, which forms a dimer having a molecular weight of about 128,000 (amorphous) or a trimer of MW 184,000 (crystalline). For light emission, molecular oxygen, a peroxide, and Fe2+ as well as the photoprotein are necessary. Moreover, two cofactors are involved; one (cofactor I) is a high-molecularweight nucleoprotein relatively stable toward heat. Another factor (cofactor II) is a kind of lipid and can be replaced with a crude hyaluronidase. The peroxide for the light emission is that of dioxan or tetrahydrofuran. Hydrogen peroxide has little effect and no effect is observed with the hydroperoxide of tetrahydropyran, diethyl ether, or cumene. Cofactor II may act to protect the photoprotein and cofactor I from nonluminescent decomposition by Fe2+ and peroxide. Quantum yields of the luminescence are 0.0093 (dimer) and 0.0143 (trimer). B. Odontosyllis Odontosyllis enopula, a polychaete annelid, is well known as "marine fireworm" and lives in Bermuda Bay. The luminescence system of this worm shows L-L reaction, which requires molecular oxygen (Harvey, 1931). Shimomura et al. (1963a) reported that the luciferin is colorless (Xmax 230, 285, and 330 nm) and nonfluorescent, but after luminescence (^max 507 nm) the product (Xmax 445 nm) has fluorescence of the same wave length as that of luminescence; thus the product could be the light emitter. Nonenzymatic oxidation of the luciferin with oxygen or with iodine gave a pink-colored substance (Xmax 260, 330, and 520 nm), which is stable toward luciferase and oxygen. These results are slightly inconsistent with those of McElroy and Seliger (1963) possibly because of different purity of the luciferin. Necessity of CN~ for luminescence is still obscure, and further investigations are necessary. C. Balanoglossus Balanoglossus, which belongs to Hemichordata, lives in the sand near shallow water. Dure and Cormier (1961) found that a cell-free extract of B.

210

Toshio Goto

biminiensis shows the L-L reaction that requires H 2 0 2 instead of 0 2 ; the luciferase is a peroxidase and can be replaced by horseradish peroxidase (Cormier and Dure, 1963; Dure and Cormier, 1963). The luciferin is also replaced by a chemiluminescent substance such as luminol. Therefore a "completely artificial bioluminescent system" can be obtained from luminol and horseradish peroxidase. It is extremely rare that the luminescent system is nonspecific and can be replaced by other substances. Since it is inhibited by Mn2+, CN", N3~, and so on, the luciferase may be a heme enzyme, but it is not inhibited by CO. In vivo bioluminescence requires molecular oxygen instead of H 2 0 2 (Harvey, 1926, 1952), thus suggesting that 0 2 is converted into H 2 0 2 in vivo prior to luminescence.

IX. LUMINOUS BACTERIA

Most luminous bacteria are of marine origin and hence cultivated in a medium containing salt in a concentration similar to seawater; optimum pH is 7.2 and rather low temperature (about 15°) are suitable. Luminescent dead fish and squid may be caused by infection with luminous bacteria. Many luminous fishes, such as Monocentris japonic us, Siphamia versicolor, and so on, use the light produced by bacteria cultivated in their photophores (symbiosis) (Haneda, 1977), as well as some bioluminescent Myopsid squids. The most common luminous bacteria are Photobacterium fisheri, P. phosphoreum, and Beneckea harveyi (Hastings and Nealson, 1977). A. Bioluminescence System Although there have been many studies of luminous bacteria, the first discoverer of the in vitro L-L reaction was Strehler (1953), who demonstrated the reaction with the extracts of Photobacterium fisheri, and found that NADH is extremely effective for enhancing luminescence. McElroy et al. (1953, 1954) found that another factor necessary for light production is FMN, which reacts with luciferase after being reduced by NADH to FMNH2. NADH dehydrogenase

NADH + H+ + FMN

—

> NAD+ + FMNH2

luciferase

FMNH2 + 0 2 + RCHO

> Products -I- hv

Thus NADH is not necessary if chemically reduced FMNH2 is used instead of FMN (Strehler et al.9 1954). Streheler and Cormier (1953; Cormier and Strehler, 1953) found a factor, KCF (kidney cortex factor), in pig kidney cortex for light enhancement. The factor was determined to be

4. Bioluminescence of Marine Organisms

211

palmitaldehyde, but other straight-chain fatty aldehydes of C7 to C18 have been found to be effective (Strehler and Cormier, 1954a; Cormier and Strehler, 1954; Hastings et al., 1966a). The bacterial luminescence requires molecular oxygen (Shapiro, 1934). Thus the luciferase, oxygen, FMNH2, and RCHO (R = AZ-C6H13 to A2-C17H35) are essential components for luminescence. With these substances, however, only a flash light is produced. For the light to last longer it is necessary to add NAD as well as crude enzyme, in which NAD is reduced by NADH-dehydrogenase to NADH, which in turn reduces FMN to FMNH2 (Duane and Hastings, 1975; Gerlo and Charlier, 1975). Although it seemed reasonable to assume that FMN is the light-emitter, the following objections arose: (i) the bioluminescence spectrum of bacteria (Xmax 480-495 nm) is far different from that of FMN fluorescence (\max 530 nm) and (ii) the reaction (FMNH2 + 0 2 —> FMN + H202) cannot produce sufficient energy for the light production (about 60 kcal/mol) and FMN is not decomposed further, since one molecule of FMN gives at least 20 photons. There have been many studies on the role of the long-chain aldehyde. Strehler (1955) assumed it to be a catalyst, but McElroy and Green (1955) suggested that light energy could be supplied by oxidation of the aldehyde to the corresponding carboxylic acid through a hydroperoxide, since the total light yield is proportional to the quantity of added aldehyde. Cormier and Totter (1957) supported McElroy's suggestion based on a study of quantum yields of this luminescence. Recently it was proved that the carboxylic acid is indeed produced directly from the aldehyde during light production (Eberhard and Hastings, 1972; McCapra and Hysert, 1973; Shimomura et al., 1972, 1974a; Vigny and Michelson, 1974). The longchain aldehyde increases light yields but has no influence on the oxidation rate of FMNH2; it participates in the reaction at a later stage than the ratedetermining step (Strehler and Cormier, 1954b). B. Bioluminescence Intermediates When FMNH2 is mixed with luciferase in the absence of oxygen, intermediate I is produced (Spudich and Hastings, 1963; Hastings et al., 1963, 1964, 1965a,b, 1966a; Gibson ef al., 1965). In the presence of oxygen, intermediate I is immediately converted to intermediate II (a peroxide), which is fairly stable; its halflife is of the order of 10-20 sec at 20° (Hastings et al., 1973a,b). Without the aldehyde intermediate II gives little light with production of FMN and H 2 0 2 , but after freezing at -77° it gives light around -3° with fairly high efficiency (Hastings et al., 1964). The reason for the freezing effect is still unknown. In the presence of aldehyde, an aldehyde-intermediate II complex (intermediate HA) is

212

Toshio Goto

formed, which can be detected by its uv spectrum at -30°; bioluminescence occurs on warming and forms FMN but no H 2 0 2 (Hastings and Balny, 1975). 02

E +FMNH2

RCHO

> E— FMNH2—-> E—FMNH 2 —0 2

> RCHO—E—FMNH 2 —0 2

I 02 FMN + H202

no RCHO E + FMN + H202

A (product)* E + FMN + hv

C. Bacterial Luciferase Bacterial luciferase has been extensively purified (McElroy and Green, 1955; Kuwabara et al., 1965; Hastings et al., 1965a; Nakamura and Matsuda, 1971). Soluble proteins in a bacterial cell consist of 2-5% luciferase (Hastings et al., 1965a; Kuwabara et al., 1965). Bacterial luciferase is a heterodimer consisting of a (MW 42,000) and ß monomers (MW 37,000) (Hastings et al., 1969; Meighen et al., 1970); the a monomer occupies the catalytic site, but the function of the ß monomer is unknown (Gunsalus-Miguel et al., 1972). FMNH2 binds with luciferase in a 1:1 ratio (Meighen and Hastings, 1971) to form nonfluorescent intermediate I. The luciferase is inactivated by blocking one sulfhydryl group in a hydrophobic environment on the α-subunit, thereby suggesting that the active center is in a region of great hydrophobicity (Nicoli and Hastings, 1974; Nicoli et al., 1974). The number of molecules necessary to produce a photon are 2800 (Cormier and Totter, 1966) or 150 (Gibson and Hastings, 1966) for NADH, 20 for the aldehyde, 0.28 or 0.34 (Cormier and Totter, 1966) for FMN (recycled), and 4 for luciferase (one cycle) (Hastings et al., 1965a); FMN is not decomposed during luminescence. Watanabe and Nakamura (1972) determined the ratio of luciferase, FMNH2, and 0 2 to be 1:1:1, although Lee (1972) and Lee and Murphy (1975) postulated sequential oxidation of two FMNH2 molecules for the formation of intermediate II. Becvar and Hastings (1975) confirmed the 1:1 ratio of luciferase and FMNH2. The overall reaction can be shown by the following equation. FMNH2 + RCHO + Oz

luciferase

> FMN + RCOOH + H 2 0 + -0.2 hv

D. Chemical Reactions for Light Emission Several mechanisms had been proposed (Eberhard and Hastings, 1972; Hastings et al., 1973a; McCapra and Hysert, 1973; Keay and Hamilton, 1975; Bruice, 1976; Lowe et al., 1976; Hemmerich, 1976), but recently

213

4. Bioluminescence of Marine Organisms

Kemal and Bruice (1976, 1977) and Kemal et al. (1977) disputed them. They suggested from model chemiluminescence studies a probable mechanism, which has some similarities to that proposed by Hastings et al. (1973a) (Scheme 8). Shannon et al. (1978) reported a deuterium isotope effect of a longchain aldehyde (1-2H) on the kinetics of light production, which suggests that scission of the aldehyde C(l)—H bond is not concerted with the formation of the emitter. The mechanism of Kemal and Bruice (1977), in which production of the emitter is concerted with the C—H bond scission, is therefore excluded. They supported the mechanism of Lowe et al. (1976), in which the excited state is produced by a 2,2-cyclore version similar to the dioxetane decomposition. They also suggested that FMN produced in the excited state may be protonated at N-l (pKa 5.2 in excited state); protonated FMN has a fluorescence spectrum similar to that of

FMNH2 II >

N*

Nv^N H

\

L

R COOH

OH

H

2N

o

+

R—C— 0

V

NH

2N

0

htf R I

o FMN Scheme 8

Toshio Goto

214

FMNHo 02

[

i

f Η

1 RCHO || T

ό°

1 /'"frAOH

OH

HO

xxncNh

ΑΛ^ΝΗ

NH

II

*'

I fn o

■ioo

1©II

S

I

0H

OH

ΝγΟ NH

0 +

J

* hi>

0 FMN

RCOOH

Scheme 9

bioluminescence (Eley et al., 1970), although this mechanism still has some difficulties. McCapra and Leeson (1976) proposed another mechanism involving intermediate II having 10a-hydroperoxide from the study of model compounds. Gast et al. (1978) showed the presence in luciferase preparations of a low-molecular-weight protein having blue fluorescence whose emission spectrum (Xmax 470 nm) exactly matches the bioluminescence spectrum; they suggested that the protein is the emitter. E. Formation of Luciferase Although luminous bacteria grow exponentially in a fresh medium, luciferase synthesis is first repressed and then, after some induction periods, activated (Nealson et al., 1970). It was suggested that the fresh medium contains a low-molecular-weight inhibitor, which is removed (Kempner and Hanson 1968), and that an activator (inducer) is produced by bacteria (Eberhard, 1972); the latter is regarded as a bacterial pheromone (Eberhard, 1972). Hastings and Nealson (1977) suggested that luminous bacteria may be nonluminous in seawater but may become luminous in photophores of fish. The bacteria produce an autoinducer;

4. Bioluminescence of Marine Organisms

215

when it surpasses an accumulated concentration in fish photophores, synthesis of luciferase is initiated. Watanabe et al. (1975), however, observed that luciferase contents remain constant during the early growth of P. phosphor eum and suggested that there is competition between the luciferase system and an electron-transfer system involving cytochromes for NAD(P)H.

X. BIOLUMINESCENT DINOFLAGELLATES

Near-surface luminescence in the oceans comes mostly from the dinoflagellates, single-cell organisms about 0.04-2 mm in diameter, such as Noctiluca and Gonyaulax, whose abnormal increase causes red tides. Gonyaulax polyedra, which has been studied most extensively, has a soluble luminescence system and a particulate one; the soluble system has luciferin and luciferase, which were found by Hastings and Sweeney (1957), whereas DeSa et al. (1963) found the particulate system that was called scintillon (Hastings et al., 1966b). A. The Luciferin-Luciferase System Bioluminescent systems for other dinoflagellates are similar to that of G. polyedra. Luciferins and luciferases can cross-react among the following dinoflagellates: G. polyedra, G. monilata (Hastings and Bode, 1961), Pyrodinium bahamense, D. lunula, Pyrocystis fusiformis, P. noctiluca (Hamman and Seliger, 1972), and Noctiluca miliaris (Eckert et al., 1965; Eckert, 1966). The soluble system containing luciferin and luciferase can be extracted with cold water, but the extract does not produce light unless salt is added at a concentration of about 1 mol/liter. The luciferase has been obtained in fairly pure form (MW 130,000) (Krieger and Hastings, 1968; Kriegerei al., 1974). The luciferin has been difficult to purify, for it is liable to be oxidized; its molecular weight is suggested to be about 400 (Hastings, 1968). Oxygen is necessary for light production (Hastings and Sweeney, 1957; Hastings et al., 1966b; Sweeney and Bouck, 1966) and the in vivo luminescence spectrum peaks at approximately 474 nm (Schmitter et al., 1976). B. Sein til Ions: Bioluminescent Particulates Scintillons can be extracted from G. polyedra, G. tamarensis, Dissodinium lunula, Pyrocystis noctiluca (Schmitter et al., 1976), Noctiluca

216

Toshio Goto

miliaris (Hastings et al., 1966b), and Pyrodinium bahamense (Fuller et al., 1972). Scintillons emit a flash of light in vitro when the pH is lowered from 8 to 5.7. They are composed of luciferase, luciferin-binding protein (LBP), and bound luciferin. LBP (MW about 120,000) is bound to the particle chiefly by electrostatic forces, whereas the attachment of lucif erase involves hydrophobic interactions, and the two are present in the particles in approximately equal amounts (Henry and Hastings, 1974). On lowering the pH, LBP releases luciferin and the luciferase becomes active, thus providing a dual control over the bioluminescence activity (Fogel and Hastings, 1972; Schmitter et al., 1976). Activity can be restored in discharged scintillons by returning the pH to 8.0 and recharging with luciferin; similar restoration is observed in the case of LBP (Fogel and Hastings, 1972; Fuller et al., 1972). C. Circadian Rhythm of Bioluminescence Bioluminescence of G. polyedra shows circadian rhythms (Sweeney and Hastings, 1957, 1958; Hastings and Sweeney, 1958) and photoinhibition. There is a diurnal variation in lucif erase activity in G. polyedra both in a light-dark cycle and in constant dim light (Hastings and Bode, 1962; McMurry and Hastings, 1972). The lucif erase and luciferin activities in the day phase G. polyedra are only 10-20% of the nighttime levels (Hastings and Bode, 1962). The same was observed in LBP activity; extracts of cells harvested in the middle of the night are five to ten times more active than extracts of cells from the middle of the day (Schmitter et al., 1976).

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