ANNUAL REVIEWS

Further

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BIOLUMINESCENCE: RECENTADVANCES

x882

Milton J. Cormier, John Lee, and John E. Wampler Bioluminescence Laboratory, Departmentof Biochemistry,University of Georgia, Athens,Georgia30602and TheUniversity of GeorgiaMarineInstitute, Sapelo Island, Georgia31327

CONTENTS INTRODUCTION........................................................... COELENTERATEBIOLUMINESCENCE......................................... The Anthozoans .............................................................. Requirements for blue light emission ............................................ Requirements fi~r green light emission ........................................... Control of the biolumineseent flash ................................................. The Hydrozoans and Ctenophores ................................................ BACTERIAL BIOLUMINESCENCE............................................. The Chemical Reaction ......................................................... The Emitting Chromophore ...................................................... FIREFLY BIOLUMINESCENCE............................................... PHOLAD BIOLUMINESCENCE AND THE PEROXIDE SYSTEMS ..................

255 256 256 257 259 260 261 263 264 266 267 268

INTRODUCTION This review is not intended as a comprehensive.onein the field of bioluminescence. A numberof topics are not mentionedbut most of these have been covered in reviews and popular articles published elsewhere (1-14). The coverage represents relatively recent advances in selected areas which the authors feel are among the more significant ones madeduring the past five years. Bioluminescenceinvolves an oxidative event, and the fact that light is a PrOductof the reaction results from the formation of a product moleculein its first electronic singlet excited state. This product moleculemaybe efficiently fluorescent or, if not, may transfer its excitation energy to an efficiently fluorescent acceptor, thus representing a case of sensitized bioluminescence. The enzymatic mechanisms involved in these oxidations appear to represent variations of the oxygenaseand peroxidaseones (2). Theterms used in this field may

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256

CORMIER, LEE & WAMPLER

(b) Luciferin: a generic term referring to a reduced compoundwhich can oxidized in an appropriate environmentto produce an electronically excited singlet state. This excited singlet state product maybe fluorescent and thus may be the emitter. Alternatively, the energy of this excited state product maybe transferred to a fluorescent acceptor which functions as the emitter. This would becomea case of sensitized bioluminescence as mentioned above. (c) Photoprotein : a protein-chromophore complexthat reacts with certain ions, such as calcium, to produce light. It is believed to be a stabilized oxygenated intermediate ofa protein-luciferin chromophorecomplex(see section on coelenterate bioluminescence). (d) Bioluminescenceor chemiluminescencequantumyield (Q~ QcL) : t herati o of the total number of photons produced to the total number of molecules of substrate utilized ; this relationship is frequently expressedas the numberof einsteins per mole. QBor QcLis the product of several terms, thus QB or QcL

= QC × QF × QEx

whereQc is the chemical yield of product, QFis the fluorescence quantumyield of the emitter, and QExis the fraction of the product moleculesproducedin an electronically excited state as opposedto the groundstate of the molecule. For somebioluminescent and chemiluminescent reactions, Qc approaches 1 and this term, therefore, can be ignored in those cases. For the theoretical organic chemists in this field, one of the central questions relates to howelectronic excited states of singlet multiplicity are efficiently generated during bioluminescence. Whereverpossible this problem is given attention in this review. Beyondthis fundamentalquestion the reader should gain an appreciation of the potential usefulness of these bioluminescent systems in the study of important biological problems, such as the control of nerve-linked calcium transients, mechanisms of flavoprotein catalysis, and mechanismsof the copper-containing peroxidases. In addition, there are numerousanalytical applications of these systems reviewed elsewhere 05, 74, 75). COELENTERATE

BIOLUMINESCENCE

Coelenterates are a diverse group of marine animals noted for their beauty and brilliant displays of bioluminescence. The molecular basis for bioluminescence has been investigated in a numberof these creatures over the past decade. These data are summarizedin this section and emphasis is placed on chemical similarities of the bioluminescent systems amongthe coelenterates. The Anthozoans The bioluminescence of several Anthozoans has been examined at the biochemical level. These include the sea pens (Stylatula, Renilla, and Acamhoptilum),the sea feather (Ptilosarcus), Parazoanthus, and Cavermdaria. Sea pansy (Renilla) bioluminescence has been studied in greater detail and consequently discussion is limited to it. However,recent data suggest that the chemical requirements for light

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257

emissionand its control are similar, if not identical, in all the Anthozoansstudied (1, 16-2S).

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REQUIREMENTS FOR BLUE LIGHT EMISSION Structural

elucidation and chemical

synthesis of a partially active analog of Renilla luciferin were achieved recently by Hori & Cormier (26). Subsequently, a fully .active luciferin (II, Figure 1) synthesized (27). Theimidazolopyrazinering systemwhichoccurs in Renilla luciferin also exists as part of the structure of several luciferins of marine origin, which include the crustacean Cypridina, a numberof fish, numerouscoelenterates, and possibly squid (1, 2, 28). Theonly differences in the structure of Renilla and Cypridina luciferins are found in the side chains, which are apparently determined by the aminoacids utilized in their synthesis. Chemicalevidenceis available for this since a numberofluciferin analogs have been prepared by condensations of any three amino acids selected to yield the desired analog(29). Synthetic Renilla luciferin, as well as native luciferin (I, Figure 1), reacts with luciferase and molecular oxygen to produce blt~ light (2v~ = 490 nm). The bioluminescence quantumyield in each case is approximately 5%(27). Theproducts of bioluminescentoxidation of Renilla luciferin are oxyluciferin (IV, Figure 1) and CO2(27, 30). Approximatelyone mole of each is produced per mole luciferin oxidized. Thecharacteristic visible absorption of luciferin at 435 nmis lost after bioluminescenceceases, and a newabsorption is found at 335 nmcharacteristic of the product oxyluciferin (27). T O~q,fR

I~"

Renillo Luciferin ~

]]

O~CH20 I ~i~

RenilloOxyluciferin (,synthetic) ~ O*C-CH2~/

H2

N~CH~ ~N~Q

Luciferin(synthetic)

C_CH2 ~"

~

Oxyluciferin Monoanion

Fiyure1 Structures of Renilla luciferin, oxyluciferin, andsomeof their derivatives. Additionally, a comparisonof the structure of synthetic Renilla oxyluciferin with Aequorea oxyluciferin. 1 2n, 2eL, and 2v refer to the wavelengthmaxima of bioluminescence,chemiluminescence, andfluorescenceemissions,respectively.

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258

CORMIER, LEE & WAMPLER

Luciferin is oxidized in aprotic solvents, such as dimethylformamide(DMF),via chemiluminescentpathway(31) to yield oxyluciferin, CO2,and a bluish luminescence (2~L = 480 nm). Oxygenis required for light production and approximately one mole of oxyluciferin is produced per mole of luciferin utilized (27). A study of this chemiluminescent reaction (27, 31) revealed that the blue emission is due to the electronic excited state of the monoanionof oxyluciferin (V, Figure 1) rather than the neutral species (IV, Figure 1) or the dianion, whichproduces yellow-greenlight. Theevidence suggests that the monoanionis formeddirectly and that its fluorescence decay rate is faster than the rate of protonation in DMF.Similar conclusions have been reached using model compounds(29, 32, 33). The electronic excited state of the monoanionof oxyluciferin (V, Figure 1) also appears to be responsible for the bluish emission observed in bioluminescence(27). Of interest here is the observation that neither oxyluciferin nor its monoanionare fluorescent in an aqueous environment (27). In DMF,however, these species are highly fluorescent both for oxyluciferin and several of its analogs (27). For example, oxyluciferin (2~- = 402 nm) has a fluorescence quantumyield of 23%in DMF,whereas its monoanion(2 e = 480 nm) should have a fluorescence quantum yield of 6~, as deduced from studies on synthetic derivatives (27). Since the bioluminescence quantumyield is 5~o and since its 2n is at 490 nm, it appears that bioluminescenceis derived from the electronic excited state of a luciferase-oxyluciferin monoanion complex. Apparently DMFand luciferase provide similar environments for

O,~---~-R~ N~N

O,~-e R~ N ~N

O2~

O-OH

e O,/..~- R ~ "~

O~’~"~

N "~’N

B ~

~ /

~-f’O

~N~N

.N~Ne LOXYLUCIFERIN ~ ~] ~ ~ONOANION

~XYLUCIFERIN ¯ LIGHT

¯Fiyure 2 Proposedalt~a(~ m~cha~isms [or the biolumincsc~uto~idation o[ ~,il~ luciferin.

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BIOLUMINESCENCE

259

fluorescence with a quantumyield of 5-6~o for the emitting species in each case. An additional reason for suggesting a QF of approximately 5~o for the luciferaseoxyluciferin complexis due to the observedfivefold increase in Qa in the presence of the green fluorescent protein (1) whoseQvis 30~(see next section). The chemiluminescence quantumyield ofluciferin in DMF,however,is only 0.1~ (27). Thus, during bioluminescenceRenilla luciferase appears to be generating electronic excited states in yields approaching 100Kas opposed to the chemiluminescent path in which the excitation yield is only 2~. Mechanismstudies using 180 labeled water and oxygen suggest the schemeshown in Figure 2 (pathway A) for the bioluminescent oxidation of luciferin (30). The labeling patterns were consistent with this schemesince oxygenin the COzproduced was derived from H20, not 02. Similar labelings were shown in studies on the bioluminescent oxidation of firefly luciferin (35). Using the same techniques and aprotic solvents, the mechanismsinvolved in the chemiluminescent oxidations of firefly luciferyl adenylate and Renilla luciferin have recently been examined.As in bioluminescence, the results are in accord with pathwayA in Figure 2 (unpublished results). Pathway B (Figure 2), which suggests a dioxetanone intermediate, attractive theoretically (36). Nevertheless,it does not appearto be involvedin Renilla and firefly luminescencesince it is inconsistent with the 180 studies. Furthermore, dioxetanonederivatives have been synthesized but their decompositionsyield triplet, not singlet excited states (37-42), and in bioluminescenceweare dealing with excited states of singlet multiplicity. Analogous1 s O studies (43) on the mechanismof the bioluminescent oxidation Cypridinaluciferin are surprising since the results suggest pathwayB (Figure 2) but the structures of Cypridinaand Renilla luciferins are similar (1). However,it is not inconceivablethat two different pathwayscould give rise to an excited state product. Further work is obviously needed to clearly understand the oxidative mechanisms involved in bioluminescence and chemiluminescence. A mechanismis required that provides a theoretical explanation for the generation of electronic excited states of singlet multiplicity and also takes into account the is O labeling patterns. REQUIREMENTS FORGREEN LIGHTEMISSION Spectral measurements on the in vitro and in vivo bioluminescence emissions in Renilla and a numberof coelenterates examinedhave shownthat the in vitro emissions (2B = 470-490nm)arc blue, whereas the in vivo emissions (2B = 509 nm) are green (19-22, 25, 44, 45). Precise spectral comparisonshave shownthat in Renilla and several other coelenterates, the green in vivo emissionis due to a highly fluorescent protein whichhas beenisolated from these animalsand is referred to as the "green fluorescent protein" (22, 25, 44, 45). In Renilla this protein (mol wt -~ 40,000) contains a bound chromophoreof unknownstructure whosefluorescent quantumyield is 30~ (1, 44). The fluorescence emission of this protein matchesprecisely the green in vivo emission (44). The possible involvementof energy transfer has been invoked to account for the differences in color betweenthe in vitro and in vivo emissions in Renilla (19, 21, 44), and subsequent studies have tended to confirm this suggestion (2, 44, 46). For example,the green fluorescent protein will not catalyze the bioluminescentoxidation

260

CORMIER,

LEE

&WAMPLER

PATHWAY TO BIOLUMINESCENCE IN THEANTHOZOANS: Binding

Protein-Luciferin

Luciferin

+ 02 + 2 Luciferase~

Lueiferase-0xyluciferin*

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+ Ca++~

Luciferase-Oxyluciferin

Binding

Protein-Ca

+ Lueiferin

Luciferase-Oxyluciferin*

+ GFP (Green

Fluorescent

Prote~n)

+ CO ~ GFP* + Luciferase-Oxyluciferin

+ Light (Blue)

GFP + Light (Green)

PATHWAY TO BIOLUMINESCENCE INTHEHYDROZOANS: Photoprotein

+ Ca++ ~

Photoprotein-Ca

Photoprotein-Ca

Protein-Oxyluciferin*

Protein-Oxyluciferin

F~ure

3

>

+ GFP

+ Light (Blue)

Protein-Oxyluciferin*

(Blue Fluorescent Protein)

GFP* + Protein-0xyluciferin

~FP + Ligh£ (Green)

A comparisonOf the bioluminescentsystems in the Anthozoansand the Hydro-

zoans. of luciferin in the absenceof luciferase, but its addition to an in vitro bioluminescent reaction will change the color of light from blue to the characteristic green in vivo emission (44, 46). Because the concentrations of luciferase and green fluorescent protein were low (~ 10-6 to 10-5 M) under conditions in which efficient energy transfer was observed, protein-protein interaction was suggested as a means of achieving a calculated critical transfer distance of 28 A (44, 46). The phenomenon is highly protein concentration dependent (46), and the quantumyield relative luciferin increases from 5%for the luciferase-catalyzed reaction to about 25~oin the presence of the green fluorescent protein (1). ,Thus, the production of both blue and green light maybe viewed as illustrated in Figure 3. The phenomenonis thought to involve nonradiative energy transfer from the electronic excited state of an oxyluciferin monoanion-luciferase complex to the chromophore on the green fluorescent protein (1). This wasthe first clear case of a "sensitized bioluminescence" (see Introduction). OF THE BIOLUMINESCENT FLASH A protein important in the control of bioluminescencein Renilla and other Anthozoanshas been recently isolated (24). It a luciferin binding protein (mol wt _~24,000), whichreversibly discharges its bound CONTROL

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BIOLUMINESCENCE261 luciferin in the presence of calcium ions and exhibits a deep yellow color when highly purified due to its associated luciferin (24). It nowappearslikely that luciferin sulfokinase, whichconverts luciferyl sulfate (III, Figure l) to luciferin in the presence of 3’,5’-diphosphoadenosine (4?), mayplay an important role in controlling the charging of the luciferin binding protein. The addition of calcium ions to a solution mixture of luciferase, oxygen, and the charged luciferin binding protein results in a bluish luminescencetypical of the in vitro reaction described above. This calcium-indmcedluminescencecan be viewedas illustrated in the upper part of Figure 3. Therate of light intensity decay in this reaction is muchslower than the rapid flash from the in vivo luminescence(1). Thus, it appears that calcium access to the luciferin binding protein in vivo must be under very fine control. An advance in understanding the mechanismsinvolved in such control was made with the discovery and isolation of lumisomesfrom Renilla by Anderson & Cormier (23). Lumisomesare membrane-boundedvesicles (average diameter = 0.2 ~tm) that produce an oxygen-dependentflash of green light (28 509 nm) whenexposed to a hypotonic solution of calcium ions (23). These vesicles contain all the proteins necessary for bioluminescence and its control, i.e. luciferase, the luciferin binding protein, and the green fluorescent protein (1, 23). All these proteins appear to be membranebound (23), providing an excellent environment for the orderly arrangement of proteins necessary for the control and energy transfer processes outlined above. A recent electron microscopystudy has revealed the location and substructural features of Renilla photocytes (48). The photocytes are highly specialized cells containing large membrane-bounded vesicles (diameter = 4-6/~m) which have been termed luminelles (48) Luminelles house hundreds of smaller vesicles which are thought to be lumisomessince they are the same size and shape as the lumisomes isolated earlier by Anderson& Cormier (23). In partially purified preparations, luminelles appear as large (4-6 #m) green fluorescent structures which produce flashes of green light whenexposed to a hypotonic solution of calcium ions (J. M. Anderson and M. J. Cormier, unpublished). Both the luminelle and lumisome membranesmay play important roles in regulating calcium transients to and from the calcium binding sites on the luciferin binding protein. Just howa nerve impulse might trigger the release of calcium in Renilla is unknown. The Hydrozoans and Ctenophores The bioluminescent systems of the jellyfish, Aequorea, and the ctenophores, Mnemiopsisand Bero~, have been the most thoroughly investigated amongthese two groups. Protein-chromophore complexes, termed photoproteins, have been isolated and react with calcium ion to produce a bluish luminescence in vitro (49-55). illustrated in Figure 3, this in vitro luminescenceis independentof dissolved oxygen. The photoprotein isolated from Aequoreahas been termed aequorin while the ones isolated from Mnemiopsis and Bero~ have been termed mnemiopsin and berovin, respectively (49, 53, 55). The protein componentsof photoproteins apparently are species-specific single polypeptide chains ranging in molecular weights from about 24,000to 31,000(54, 56).

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262

CORMIER~ LEE & WAMPLER

Cormier and associates (1, 25-27, 57) have provided evidence that the native chromophorecomponentof photoproteins is similar, if not identical, to Renilla luciferin, although Johnsonand associates (3, 58) object to this idea. The suggestion that the native chromophores are similar is strengthened by the finding of Shimomura& Johnson (58) that the aequorin bioluminescence reaction forms product isolated and identified as VI in Figure 1. Wehave labeled it Aequorea oxyluciferin because of its near identity with Renilla oxyluciferin (IV, Figure 1). The emitter during aequorin bioluminescenceis a prote.in-oxyluciferin complex(58), again analogous to Renilla bioluminescence. Luciferyl sulfates, indistinguishable from Renilla luciferyl sulfate (III, Figure 1), have also been isolated from numerous coelenterates including Aequoreaand Mnemiopsis(17, 25). Furthermore, the visible absorption bands of aequorin (1, 59) and mnemiopsin(54) can now be explained the basis of spectral perturbations of a Renilla-like luciferin chromophore(1, 57). Finally, an oxidative path similar to that illustrated in Figure 2 is a reasonable possibility for the formation of Aequoreaoxyluciferin (VI, Figure 1). Apparently,the oxygen-requiring step has previously been incorporated into these photoproteins, resulting in an oxygenatedintermediate that is stable in the absence of calcium. The nature of this intermediate and the lack of CO 2 production during the bioluminescence 2reaction of aequorin (60) remains to be explained. Whereas the chemical

paths

to light

emission

in Renilla

and Aequorea (and perhaps

ctenophores)appear to be similar, there are basic differences reflected at the protein level in control of the luminescencein each case. Asshownin Figure 3, two proteins, a luciferin binding protein and luciferase, are involved in calcium-induced biolumirkescencein the Anthozoans,whereasonly a single protein, a photoprotein, is involved in the Hydrozoansand ctenophores. Photoproteins appear to be playing a dual role; they apparently bind a Renilla-like luciferin chromophorein the absence of calcium ions thus acting like a binding protein, while converting this chromophore to oxyluciferin in the presence of calcium ions thus acting like a luciferase. Like the Anthozoans, the bioluminescent Hydrozoans that have been examined contain a green fluorescent protein (21, 25, 45, 49, 61). The fluorescence emission the green fluorescent protein isolated from Aequoreais identical with the one isolated fromRenilla (25, 45), althoughtheir excitation spectra are quite different (1, 45). suggests differences in either the proteins, the chromophores, or both. Since the in vivo emission is green ().s = 509 nm) as opposed to the blue in vitro emission aequorin (,~oB -~ 469 nm), a sensitization by the process of energy transfer, such proposedfor Renilla, has beensuggested as an explana, tion (2, 21, 45, 62). Using purified preparations of the Aequoreagreen fluorescent protein, which contained six green fluorescent protein components,a sensitization of the in vitro reaction by energy transfer has been demonstrated (45). However,it is difficult suggest a mechanismfor this sensitization because of the reported quantumyield data. Efficient nonradiative energy transfer should give an increase in quantumyield 2 Since completionof this manuscript,a paper by Shimomura et al demonstratesthat CO~ is producedduring the calcium-inducedbioluminescenceof aequorin (Biochemistry1974. 13:3278). Furthermore, they present data which can be explained by the presence of Renilla-like luciferin chromophore in aequorin.

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BIOLUMINESCENCE

263

if the acceptor (green fluorescent protein) has a significantly higher quantumyield than the donor(oxylucifcrin-protcin complexexcited state). Since the bioluminescence quantumyield of aequorin alone is 23~, and the fluorescence quantumyield of the product (oxyluciferin-protein complex)is probably not muchgreater than 23~ (45), an increase in quantumyield in the presence of green fluorescent protein (Qv = 72~) would be expected. However, the bioluminescence quantum yield was only 23~ under conditions of efficient energy transfer (45). The regulation of calcium transients to the photoproteins in vivo probably occurs in a manneranalogous to that described above for the Anthozoans,since Renilla-like lumisomeshave been isolated from several species of bioluminescent Hydrozoans (1, 23). The Hydrozoanlumisomescontain a photoprotein and a green fluorescent protein but no luciferase or luciferin binding protein. BACTERIAL

BIOLUMINESCENCE

The bioluminescenceof marine bacteria has been studied mostly on purified extracts and consequentlya lot moreis knownabout the in vitro reaction than the in vivo one. The in vitro reaction is a luciferase-catalyzed, molecular oxygen oxidation of reduced flavin mononucleotide (FMNH2)and an aliphatic aldehyde (RCHO) a carbon chain length longer than heptanal (63-66). It is likely that free FMNHz not available within the cell and, in fact, what appear to be flavoproteins that have activity in the light reaction havebeenisolated fromcell extracts (67, 68). Thereis yet no strong evidence for RCHO involvement in vivo (69, 70). It was first thought that reduced nicotine adenine dinucleotide (NADH) was the substrate for the reaction (71), but further purification of the enzymeremovedthis activity and it was then shown that the true substrate was FMNHz (63). NADH probably reduces FMNin the crude preparation via an FMN-linked NADH dehydrogenase. Advantage can be taken of this dehydrogenase in applying the bacterial bioluminescence reaction to analytical work, since manyenzymescan be readily coupled to NADH (72-75). Thetotal light producedin the reaction is proportional to the amountof each of the substrates (02, FMNH2, and RCHO)when they are present in limiting quantities (63-66). The same can be said of the lucifer~se since excess FMNH2 is autooxidized so rapidly in comparisonto the rate of light emissionthat it is gone whenthe luciferase finishes its catalytic cycle (76). Theluciferase, therefore, acts only oncein the in vitro reaction like the other reactants, but it appears capable of turnover on repeated addition of FMNH2 (77, 78). Although the role of RCHO was not understood for a long time (7, 79), it is nowestablished that it is a true substrate and oxidized to the corresponding carboxylic acid (80-83), as originally suggested McElroy& Green (84). Characteristics of the bacterial reaction, such as kinetics, absolute quantumyields, and emission spectra, vary with the type of bacterium from which the luciferase is purified (11, 68, 85, 86). Extensive studies of the luminous bacteria taxonomyare recently available (87-89), and mutants with altered reaction and spectral properties have been demonstrated(90, 142). Thefact that this luciferase is of bacterial origin

264 ’ CORMIER,LEE & WAMPLER means that large quantities may be produced by fermentation and purified by relatively straightforward methods(76, 91, 92).

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The Chemical Reaction Luciferase is a protein of molecular weight about 80,000 (86) and contains no metals or other cofactors (76, 91, 93). It maybe reversibly dissociated into two subunits whichdiffer slightly in molecularweights(86, 93, 94). The first step in the reaction is a one-to-one association of FMNH2 with the luciferase (E) with an equilibrium constant of 3 × 104 M1 at 23°C (95, 96). As 3 also increases concentration of E increases above that of FMNH 2, the QB(FMNH2) and reaches a constant value when E > 30~M(for FMNH2> 16pM) (97). results from a competition between autooxidation and E for the available FMNH~ o2 FMNH2+ E~ E- FMNH2------~

X

FMN + H202 whereX represents intermediates in the light path. Stopped-flowobservations show no contribution to the rate of FMNappearance from the more rapid auto-oxidation when E > 30/~M (96). Twoapproaches to determining the reaction stoichiometry have been made, the ~st a direct measurement of substratc utilization (97) and the secondby stead~/-state kinetic analysis (98, 99). Using a luciferasc concentration of greater than 30 ~M eliminate auto-oxidation, the utilization of each substrate determinedon the basis of its quantumyield gives 2FMNH~+ 202 + RCHO~ 2FMN+ H~O~ + [H~O] + RCOOH Both H20~and RCOOH have been identified as products (80-83, 97) but not H~O, indicated by the square brackets. A certain ambiguityexists in this approachin that, in commonwith many hydroxylase enzymes, bound FMNH2might have a certain probability of reacting in an alternate pathwaythat does not lead to light production. In the hydroxylases this is evidenced by the variation in utilization ratios whenthe substrate concentrations are varied (100, 101). With luciferase this does not happen and neither is there any change with variation of pH(6-8.5), temperature (0-35°C), type of bacteria fromwhichthe luciferase is isolated, or the numberof carbons in the aldehyde from C7 to C14(96, 97, 102). An apparently different conclusion results from the kinetic analysis. The initial steady-state rate of light emission is first order in both FMNH2 and luciferase over a broad concentration range (98, 99, 103). Thestationary state analysis has a formal relationship to reaction stoichiometry, and with this approach Meighen& Hastings (98) and Watanabe& Nakamura(99) have concluded that the reaction involves flavin instcad of two. 3 QB(FMNH2) refers to the bioluminescencequantumyield relative to FMNHz, while Qn(X2)is that relative to the intermediateX2,etc. SeeIntroduction.

BIOLUMINESCENCE265

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Clearly then, neither a one flavin nor a two flavin simple reaction scheme adequately accounts for the data. Lee & Murphy(96), therefore, propose a more complex scheme which requires two moles of flavin per mole of enzymebut which could still give overall first order kinetics. The oxidation of FMNH2 and the utilization of oxygenvia luciferase (E > 30 #M)are both biphasic in rate (96, 104). Although Yoshida et al (105, 106) noted a biphasic oxidation of FMNH2, significant contribution fromauto-oxidationis evident in their data, since insufficient luciferase was present to outcompetethis. Fromstudies of the transient kinetics of the reaction at 5°C with luciferase from the bacterium Beneckea harveyi, Lee & Murphy(104) proposed the following sequence of events 2FMNH2+ E+O2 ~ X1 +FMN+H202

fast

tl/2 ~ 1.3 min 2X1 +02-’~’X ,where X2 and X1 are viewed as the cquivalcnt of EH202 FMNand EH2FMN respectively. The first step is complete in 30 sec, at which time all the FMNis completely oxidized. If X2is allowed to stand around it eventually decays (tl/2 ~ 7 min) to FMNand H202with the release of native luciferase. If RCHO is added in excess over X2, even whenits concentration is rate limiting, thc full quantumyield of X2is 3 X2is believed to be the same as the long lived intermediate equal to 2QB(FMNH2). (II) of Hastings &Gibson(107). Hastings et al (108) attempted to separate this the reaction mixture by low temperature chromatography, but they allowed only a few seconds for oxygen uptake at 5°C before quenching to low temperature (- 20°C) in an ethylene glycol-water mixture, and it was not established that the separated intermediate did not continue to react with oxygen before reacting with RCHO. Indeed the absorption spectrum presented for intermediate II appears to have a contribution from FMNH~. Murphyet al (109) prepared Xz by converting luciferase quantitatively at 0°C where tl/2 ~ 14 min. The reaction mixture was then subjected to rapid chromatography on a short G-25 Sephadex column. They found hardly detectable amounts of FMNin the chromatographedpreparation even after warming, yet the preparation reacted with RCHO to give the same Q~(X2) as for the complete reaction in the presence of FMN.Based on the trace FMNdetectable in the chromatOgraphed X2, the QB(FMN) was shownto be greater than unity. Therefore, z t hat s eems to be a flavoprotein can be rapidly dissociated apo-X2 ÷FMN~

X 2

and the apo-X2 is separated by chromatography. Aswith most flavoproteins, FMNfluorescence is quenchedwhen bound in holo-Xz. The fluorescence quenching was used to estimate the equilibrium binding constant as 7 × 105 M-1 at 0°C (109).. The light reaction proceeds by X2 + RCHO-~ FMN+ RCOOH + H20 -k E + light In the complete absence of FMNthe light is quenched. Since only a trace of FMNis

266

CORMIER, LEE & WAMPLER

required for the full quantumyield, the FMNmust recycle in what is apparently another example of a sensitized bioluminescencereaction.

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The Emittin,q

Chromophore

It is generally believed that the emitting chromophoreis some form of the FMN molecule, although the only basis for this is an observed 20 nm blueshift in 1,~B when iso-FMNH2is used to initiate the reaction rather than FMNH2 (110). The radiative transition is almost certainly of singlet character since the presence of molecular oxygen in the reaction would completely quench a triplet, even if the chromophorewas buried in the protein (111). That the emitting chromophoreis FMNitself is excluded by the facts that isoFMNhas its 2F1 redshifted over FMNand that there is a distinct difference laetween the 2F for FMN(535 nm) and the species-dependent bioluminescence emission maxima(2~), which covers the range 478-505 nm (11, 85, 110). A perturbation FMNfluorescence is unlikely since most flavoproteins either quenchthis fluorescence or, if they are fluorescent, are ve.ry slightly blueshifted over free FMN (143). Of course, luciferase could be unique in providing a strongly perturbing environment for FMN, but it is found that even strongly perturbing solvent conditions do not blueshift the fluorescence more than about 25 nm and, more important, such a shift is accompanied by a markedvibronic splitting (102, 112, 113). In contrast, the bioluminescence spectra are all smoothstructureless curves (102). Eley et al (114) suggested that the emitter is a luciferase-bound flavin cation, ÷, based on the similarity in fluorescence emission spectra of FMNH + and FMNH ÷ iso-FMNH in rigid solvents to the bioluminescence spectra produced from FMNH2 and iso-FMNH2,respectively. Althoughthis is still a reasonable suggestion, recent experiments make the requirement that the emitter be some form of FMNless compelling (109). From what we now., know, a reaction product of FMNHz formed in only trace amounts could conceivably sensitize the process; for example, degradation of FMNto a bluer fluorescing product, such as lumichrome, occurs with a yield less than 0.02, based on quantumyield measurements(115). Furthermore, interaction of FMNwith luciferase forming a new chromophore, which was thought + (95, 114), more likely to have absorption and fluorescence properties of FMNH arises from a small amount of light-induced reduction of FMNand subsequent turnover of luciferase to form some X2, whose typical flavoprotein absorption spectrumwouldgive the sameeffect (104, 109). McCapra& Hysert (82) suggested that the emitting chromophore is a complex ofaldehydeand flavin with the isoalloxazine ring ruptured at the 2,3 position. Model compounds have fluorescence spectra similar to the bioluminescence. The mechanismproposed for its formation is incorrect, however, since it requires RCHO addition to FMNH2, and the flavin is fully oxidized in Xz (104). The identity of the emitting chromophore, therefore, still remains unknown, ÷ remains a good candidate, and a mechanismfor its formation by although FMNH one electron transfer from acyloxy radical provides a sufficiently et~ergetic and physically realistic excitation process (102)

BIOLUMINESCENCE

O

267

O

FH. +RC-O.--FH + +RC- O- +70 kcal

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FIREFLY

BIOLUMINESCENCE

Advances in the understanding of firefly bioluminescence have been reviewed by McElroy&DeLuca(116) and Cormier et al (2). The chemiluminescencereactions firefly luciferin and its analogs havebeen reviewedby Whiteet al (117). Theefficient firefly reaction (34) involves a two step conversion of luciferin, VII (Figure 4) to luciferyl adenylate, VIII (118) follow6d by oxidation to oxyluciferin, IX (119). Theconversion to luciferyl adenylate occurs via a reversible reaction (120) as follows 2 + ~ E(LHz-AMP)+ P-P E + LH2 + ATP-Mg 1. whereE is luciferase, LH 2 is luciferin; P-P is pyrophosphate, and the parentheses indicate an enzyme-boundproduct. The subsequent reaction of the enzyme-bound luciferyl adenylate involves several postulated steps as indicated by the following reaction scheme + E(LHz-AMP) ~ E(LH--AMP)+H 2. E(LH--AMP) ~ E’(LH --AMP) E’(LH--AMP) + O2

3.

ri + E(LHOO--AMP) or E(LHOOH-AMP)

E(LHOO--AMP)or E(LHOOH-AMP) ~ E(oxyluciferin

dianion

4. AMP)* 5.

E(oxyluciferin dianion-AMP)*-~ E(oxyluciferin, AMP)+hv

6.

"S’~ O

"HO"~-~’~ $

VII R=OH VIII R=AMP

ix

Figure4 Structuresof firefly luciferin (VII), luciferyl adenylate(VIII), oxyluciferin(IX), and the oxyluciferindianion(X).

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268

CORMIER, LEE & WAMPLER

The risetime of luminescencein the firefly in vitro reaction is slow (half risetime, 0.2 sec) (121). A recent investigation of the fast kinetics of this step by DeLuca l~lcElroy (122) reveals at least two slow steps, both occurring after formation luciferyl adenylate. Both these steps (2 and 3) appear to be intramolecular changes occurring before the addition of oxygen, since preincubation of luciferin, enzyme, 2+ anaerobically gives a rapid flash upon the introduction of oxygen. ATP, and Mg Initiating the light reaction with preformed LH2-AMP gives the slow kinetics. Binding of substrate to enzymedoes not account for these slow kinetics, since the kinetics are not dependent on enzyme concentration and the spectral changes occurring during binding of a substrate analog dehydroluciferyl adenylate are rapid. Based on this evidence DeLuca& McElroy(122) postulate steps 2 and 3 as changes in the enzyme-luciferyl adenylate complex; first an abstraction of a proton from luciferyl adenylate to form the carbanion for subsequent attack on oxygen and then conformational changes in the enzymecomplex. Details of the mechanismthat generates the excited state product (reactions and 5) are not clear. Twomechanismshave been postulated (35, 123) similar those suggested for Renilla (30) and Cypridina (43). As mentioned, 1802 labeling studies favor the Renilla-like mechanism. Evidence from manychemiluminescencestudies predicts emission (step 6) from the dianion (X) of the oxyluciferin product (117). Thestructure of oxyluciferin has confirmed by synthesis (124) and by purification from spent chemiluminescenceand bioluminescence reactions (119). The monoanionfluoresces red and accounts for the red emission of the in vitro bioluminescence at acid pHs. In reaction 6, AMPis shownstill bound to luciferase. This is strongly suggested by the effect of two ATP analogs, 3-iso-ATP (125) and 1,N6-etheno-ATP(126), on the reaction. 3-iso-ATP reacts with luciferin to give red light. 1,N6-etheno-ATP does not react with luciferin, but if the adenylate analog is formed chemically it is a substrate for red bioluminescence. Since the phenol hydroxyl of oxyluciferin appears to be ionized even in the ground state (117, 127), these color changesmaybe due to competition in the excited state betweenemission and either enol-keto tautomerization or protonation of the enolate ion. PHOLAD BIOLUMINESCENCE PEROXIDE SYSTEMS

AND THE

The Pholas dactylus (clam) bioluminescence system, studied by Michelson and coworkers, involves the oxidation of a protein-bound luciferin (50,000 mol wt) molecularoxygen(128). This luciferin differs from photoproteins requiring a separate enzyme(310,000 mol wt) for the reaction to occur (129). Recent evidence suggests that Pholad luciferase is a copper glycoprotein (130). The in vitro reaction stimulated by a variety of chemical agents : ferrous ion and FMNH2 (128), pyrogallol, ascorbic acid, dihydroxyfumaric acid, and catechol (129). Eachof these effects can be explained by a mechanisminvolving attack on luciferin by superoxide ions produced enzymically and by contributions to propagation of the light reaction by radical chain reaction mechanisms(129). In fact, an aerobic solution of iron II

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BIOLUMINESCENCE269 FMNH2 will cause Pholad luciferin to chemiluminesce. A detailed investigation of these reactions and other metal-catalyzed oxidations of Pholadluciferin and luminol support a mechanismfor both chemi- and bioluminescence involving primary attack on luciferin by superoxide ion and a radical chain reaction in the presence of high concentrations of certain reducing agents (131, 132). Additional support for. this mechanismis demonstrated by the fact that horseradish peroxidase can substitute for luciferase in producing bioluminescencewith molecularoxygen(133). Conversely, Pholad luciferase can serve as a peroxidase in the oxidation of several typical peroxidase substrates (133). This is not necessarily unusual since other copper proteins react with peroxide (134, 135). Someinteresting comparisons can be drawn between the Pholas system and the few peroxide-requiring bioluminescence systems so far investigated. For instance, the Chaetopterus variopedatus photoprotein requires only ferrous ion, oxygen, and a peroxide for light (136), similar to the ferrous ion-catalyzed chemiluminescence Pholadluciferin. The system from the marine worm,Balanoglossis biminiensis, crossreacts with the model bioluminescencesystem of horseradish peroxidase and luminol (137). Andfinally, luciferase from the luminescent earthworm, Diplocardia Ionga, maybe a copper protein (138, 139). Do these similarities arise from similarities in the mechanismof these reactions? A metal cofactor has not been shownfor the Balanoglossus system but certainly is indicated for the other systems, and cyanide and azide do inhibit Balanoglossidluciferase (140). By analogy to the Pholas system, a radical mechanismmight then be expected. The Balanoglossid reaction is inhibited by cysteine and mercaptoethanol, both of whichcan act as radical scavengers (137). However,the Diplocardia system does not crossreact with the others and does not oxidize typical peroxidase substrates (141). Literature Cited 1. Cormier,M. J., Hori, K., Anderson,J. M. 1974.Rely. Bioenerg.346:137-64 2. Cormier,M.J., Wampler, J. E., Hori, K. 1973. Progr. Chem.Org. Natur. Prod. 30: 1-54 3. Johnson, F. H., Shimomura,O. 1972. Photophysiology7 : 275-334 4. McElroy, W. D., Seliger, H. 1969. Photochem.Photobiol. 10:153-70 5. Hastings,J. W.1968.Ann.Rev. Biochem. 37 : 597-630 6. Goto, T., Kishl, Y. 1968. Angew.Chem. 7 : 407-14 7. Cormier, M. J., Totter, J. R. 1968. Photophysiology 4 : 315-53 8. Goto, T. 1968. Pure Appl. Chem.17: 421-41 9. McCapra,F. 1973. Endeavour32:139-45 10. Adam,W.1973. Chem.Unserer Zeit 6: 182-91 11. Seliger, H. H., Morton, R. A. 1968. Photophysiology4:253-314 12. Airth, R., Foerster,G.E., Hinde,R. 1970.

Photobiologyof Microorganisms, ed. Per Halldal, 479-94. NewYork: WileyInterscience 13. Cormier, M. J. 1974. Natur. Hist. 83: 26-34 14. Lee, J. 1974.Photochem. Photobiol.20: 535-39 15. Seliger, H. H. 1973. Chemiluminescence and Bioluminescence, ed. M.J. Cormier, D. M. Hercules, J. Lee, 461-78. New York : Plenum 16. Cormier, M.J., Eckroade,C. B. 1962. Biochim.Biophys.Acta 64 : 340-44 17. Hori, K., Nakano,Y., Cormier, M. J. 1972.Biochim.Biophys.Acta256: 638-44 18. Cormier,M.J., Crane,J. M., Nakano,Y. 1967. Biochem.Biophys. Res. Commun. 29 : 747-53 19. Hastings,J. W.,Morin,J. G.1969.Biol. Bull. 137:402 20. Morin,J. G., Hastings,J. W.1971.J. Cell. Physiol.77 : 305-12 21. Morin,J. G., Hastings,J. W.1971.J. Cell.

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Physiol. 77:313-18 22. Wampler,J. E., Karkhanis, Y. D., Mofin, J. G., Cormier, M. J. 1973. Biochim. Biophys. Acta 314:104-9 23. Anderson, J. M., Cormier, M. J. 1973. J. Biol. Chem. 248:2937-43 24. Anderson, J. M., Charbonneau, H., Cormier, M. J. 1974. Biochemistry 13: 1195-1201 25. Cormier, M. J., Hori, K., Karkhanis, Y. D., Anderson, J. M., Wampler, J. E., Morin, J. G., Hastings, J. W. 1973. J. Cell. Physiol. 81:291-98 26. Hori, K., Cormier, M. J. 1973. Proc. Nat. Acad. Sci. USA70:120-23 27. Hori, K., Wampler,J. E., Matthews, J. C., Cormier, M. J. 1973. Biochemistry 12:4463-69 28. Goto, T., lio, H., Inoue, S., Kakoi, H. 1974. TetrahedronLett. 26 : 2321-24 29. McCapra,F., Manning,M. J. 1973. Chem. Commun. 467-68 30. DeLuca, M., Dempsey, M. E., Hori, K., Wampler, J. E., Cormier, M. J. 1971. Proc. Nat. Acad. Sci. USA 68:1658~0 31. Hori, K., Wampler,J. E., Cormier, M. J. 1973. Chem. Commun.492-94 32. McCapra, F, Chang, Y. C. 1967. Chem. Commun.1011-12. 33. Goto, T. 1973. Tetrahedron Lett. 29: 2035-39 34. Seliger, H. H., McElroy, W. D. 1960. Arch. Biochem. Biophys. 88:136-4l 35. DeLuca, M., Dempsey, M. E. 1970. Biochem.Bio phys. R es. C ommun.40 : 11722 36. McCapra, F. 1968. Chem. Commun.15556 37. Kopecky, K. R., Mumford, C. 1969. Can. J. Chem. 47:709-11 38. White, E. H., Wiecko, J., Wei, C. C. 1970. J. Am. Chem. Soc. 92:2167-68 39. Lee, D. C-S., Wilson, T. See Ref. 15, 265-83 40. Adam, W., Liu, J. C., Simpson, G., Steinmetzer, H. C. See Ref. 15, 493-94 41. Turro, N. J., Lechtken, P. 1972. J. Am. Chem. Soc. 94 : 288~88 42. Turro, N. J., Lechtken, P. 1973. J. Am. Chem.Soc. 95 : 264-66 43. Shimomura, O., Johnson, F. H. 1971. Bioche~n. Biophys. Res. Commun.44: 340~46 44. Wampler, J. E., Hori, K., Lee, J. W., Cormier, M. J. 1971. Biochemistry 10: 2903-10 45. Morise, H., Shimomura,O., Johnson, F. H., Winant, J. 1974. Biochemistry 13: 2656-62 46. Wampler,J. E., Karkhanis, Y. D., Hori, K., Cormier, M. J. 1972. Fed. Proc. 31:419

47. Cormier, M. J., Hori, K., Karkhanis, Y. D. 1970. Biochemistry 9 : 1184-90 48. Spurlock, B. O., Cormier, M. J. 1975. J. Cell Biol. 64 : 15-28 49. Shimomura,O., Johnson, F. H., Saiga, Y. 1962. J. Cell. Comp.Physiol. 59:223-40 50. Shimomura, O., Johnson, F. H., Saiga, Y. 1963. J. Cell. Comp.Physiol. 62 : 1-8 51. Shimomura, O., Johnson, F. H., Saiga, Y. 1963. J. Cell. Comp.Physiol. 62:9-16 52. Ward, W. W., Seliger, H. H. 1973. Fed. Proe. 32 : 661 53. Girsch, S. J., Hastings, J. W. 1973. Am. Soc. Photobiol. 183 54. Ward, W. W., Seliger, H. H. 1974. Biochemistry 13 : 1500-10 55. Ward, W. W., Seliger, H. H. 1974. Biochemistry 13 : 1491-99 56. Kohama, Y., Shimomura, O., Johnson, F. H. 1971. Biochemistry 10:4149-52 57. Hori, K., Ward, W. W., Anderson, J. M., Cormier, M. J. 1974. Am. Soe. Photobiol. 57 58. Shimomura, O., Johnson, F. H. 1973. Tetrahedron Lett. 31:2963-66 59. Shimomura, O., Johnson, F. H. 1969. Biochemistry 8 : 3991-97 60. Shimomura,O., Johnson, F. H. See Ref. 15, 337-45 61. Morin, J. G., Reynolds, G. T. 1970. Biol. Bull. Mar. Biol. Lab. WoodsHole 139: 430-31 62. Johnson, F. H. 1967. Comp. Biochem. 27 : 79-136 63. McElroy, W. D., Hastings, J. W., Coulombre,J., Sonnefeld, V. 1953. Arch. Biochem. Biophys. 46 : 399-416 64. Cormier, M. J., Strehler, B. L. 1953. J. Am. Chem.Soc. 75 : 4864 65. Strehler, B. L., Cormier, M. J. 1954. J. Biol. Chem.211 : 213-25 66. Hastings, J. W., Spudich, J., Malnic, G. 1963. J. Biol. Chem. 238:3101~5 67. Mitchell, G. W., Hastings, J. W. 1970. Biochemistry 9 : 2699-2708 68. Lee, J., Murphy, C. L., Faini, G., Baucom,T. L. 1974. Liquid Scintillation Countiny: Recent Developments, ed. P. Stanley, B. Scoggins, 403-21. NewYork : Academic 69. Eberhard, A., Rouser, G. 1971. Lipids 6:410-15 70. Ferrell, W.J., Kessler, R. J., Drouillard, M. 1971. Chem. Phys. Lipids 6:131-34 71. Strehler, B. L. 1953. Arch. Biochem. Biophys. 43 : 67-80 72. Stanley, P. E. 1971. Anal. Biochem. 39: 441-53 73. Brolin, S. E., Borglund, E., Tegnei, L., Wettermark, G. 1971. Anal. Biochem. 42:124-35 74. Schram, E. See Ref. 68, 383-403

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BIOLUMINESCENCE 75. Stanley, P. 1974. Liquid Scintillation Counting, ed. M. A. Crook, P. Johnson. London : Heyden and Son 76. Hastings, J. W., Riley, W. H., Massa, J. 1965. J. Biol. Chem,240:1473-81 77. Erlanger, B. F., Isambert, M. F., Michelson, A. M. 1970. Biochem. Biophys. Res. Commun.40 : 70-76 78. Lee, J. See Ref. 15, 386 79. Hastings, J. W. 1968. Ann. Rev. Biochem. 37 : 592631 80. Shimomura, O., Johnson, F. H., Kohama,Y. 1972. Proc. Nat. Acad. Sci. USA69 : 2086-89 81. Dunn, D. K., Michaliszyn, G. A., Bogacki, l. G., Meighen, E. A. 1973. Biocl~emistry 12 : 4911-18 82. McCapra, F., Hysert, D. W. 1973. Biochem.Biophys. Res. Commun.52 : 298304 83. Vigny, A., Michelson, A. M. 1974. Biochemie 56:171-76 84. McElroy, W. D., Green, A. 1955. Arch. Biochem.Biophys. 56 : 24(~55 85. Seliger, H..H., McElroy, W. D. 1965. Light: Physical and Biological Action. NewYork : Academic 86. Hastings, J. W., Weber,K., Friedland, J., Eberhard, A., Mitchell, G. W., Gunsalus, A. 1969. Biochemistry 8:4681-89 87. Hendrie, M. S., Hodgkiss, W., Shewan, J. M. 1970. J. Gen. Microbiol. 64: 15169 88. Chumakova, R. I., Vanyushin, B. F., Kokurina, N. A., Vorob’eva, T. I., Medvedeva, S. E. 1973. Microbiology 41 : 53%46 89. Reicheit, J. L., Baumann,P. 1973. Arch. Mikrobiol. 94 : 283-330 90. Cline, T. W., Hastings, J. W. 1972. Biochemistry 11 : 3359-70 91. Kuwabara,S., Cormier, M. J., Dure, L. S., Kreiss, P., Pfuderer, P. 1965. Proc. Nat. Acad. Sci. USA53 : 822-28 92. Gunsalus-Miguel, A., Meighen, E. A., Ziegler-Nicoli, M., Nealson, K., Hastings, J. W. 1972. J. Biol. Chem. 247 : 398-404 93. Cormier, M. J., Kuwabara, S. 1965. Photochem.Photobiol. 4 : 1217-25 94. Friedland, J., Hastings, J. W. 1967. Proc. Nat. Acad. Sci. USA58 : 2336-42 95. Lee, J., Murphy,C. L. 1973. Biophys. J. 13 : 274a 96. Lee, J., Murphy, C. L. 1975. Biochemistry. In press 97. Lee, J. 1972. Biochemistry11 : 3350-60 98. Meighen, E. A., Hastings, J. W. 1971. d. BioL Chem. 246 : 766(~74 99. Watanabe, T., Nakamura, T. 1972. J. Biochem. 72 : 647-53 100. Walsh, C. T., Schonbrunn, A., Abeles,

271

R. H. 1971. J. Biol. Chem. 246:6855 67 101. White-Stevens, R. H., Kamin, H., Gibson, Q. H. 1972. 9". Biol. Chem. 247: 2371-82 102. Lee, J., Murphy, C. L. Scc.Ref. 15, 381-86 103. Chappelle, E. W., Picciolo, G. L., Altland, R. H. 1967. Biochem. Med. 1 : 252-60 104. Lee, J., Murphy,C. L. 1973. Biochem. Biophys. Res. Commun.53:157-63 105. Yoshida, K., Takahashi, M., Nakamura, T. 1973. Biochem. Biophys. Res Commun.42 : 1470-74 106. Yoshida, K., Takahashi, M., Nakamura, T. 1974. J. Biochem. 75:583-90 107. Hastings, J. W., Gib~on, Q. H. 1963. J. Biol. Chem. 238:2537-54 108. Hastings, J. W., Balny, C., Le Peuch, C., Douzou, P. 1973. Proc. Nat. Acad. Sci. USA70 : 3468-72 109. Murphy,C. L., Faini, G., Lee, J. 1974. Biochem. Biophys. Res. Commun.58: 119-25 110. Mitchell, G. W., Hastings, J. W. 1969. d. Biol. Chem. 244:2572-76 111. Lakowicz, J R., Weber, G. 1973. Biochemistry 12 : 4171-80 112. Palmer, G., Massey, V. 1968. Biological Oxidations, ed. T. P. Singer, 263-98. NewYork : Interscience 113. Sun, M., Moore, T. A., Song, P. S. 1972. J. Am. Chem. Soc. 94:1730-40 114. Eley, M., Lee, J., Lhoste, J-M., Lee, C. Y,, Cormier, M. J., Hemmerich,P. 1970. Biochemistry 9 : 2902-8 115. Lee, J., Seliger, H. H. 1965. Photochem. Photobiol. 4:1015-48 116. McElroy, W. D., DeLuca, M. See Ref. 15, 285-311 117. White, E. H., Rapaport, E., Seliger, H. H., Hopkins, T. A. 1971. Bioorg. Chem. 1:92-122 118. Seliger, H. H., McElroy, W. D., White, E. H., Field, G. F. 1961. Proc. Nat. Acad. Sci. USA 47:1129-34 119. Suzuki, N., Goto, T. 1972. Tetrahedron 28 : 4075-82 120. Rhodes, W. C., McElroy, W. D. 1958. J. Biol. Chem. 233:1528-37 121 McElroy, W. D., Seliger, H. H. 1961. Light and Life, ed. W. D. McElroy, B. Glass, 219-57. Baltimore: Johns Hopkins Univ. Press 122. DeLuca, M., McElroy, W. D. 1974. Biochemistry 13 : 921-25 123. Hopkins, T. A., Scliger, H. H., Whitc, E. H., Case, M. W. 1967. J. Am. Chem. Soc. 89 : 7148-50 124. Suzuki, N., Goto, T. 1971. Tetrahedron Lett. 22 : 2021-24

272

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125. McElroy, W. D., Scliger, H. H. 1966. Molecular Architecture in Cell Physiology, ed. O. Hayashi, A. SzentGyorgi, 63-79. EnglewoodCliffs, N J: Prentice-Hall 126. DeLuca, M., Leonard, N. J., Gates, B. J., Sci. McElroy, D. 1973. Proc. Nat. Acad. USAW. 70:1664-66

son, A. M. 1973. Biochimie 55:83 93 134. Jolley, R. L., Evans, L. H., Makino, N., Mason, H. S. 1974. J. Biol. Chem. 249 : 335-45 135, Felsenfeld, G., Printz, M. P. 1959. J. Am. Chem. Soc. 81:6259-64 136. Shimomura, O., Johnson, F. H. 1968. Science 159 : 1239~40 127, Bowie, L. J., Irwin, R., Loken, M., 137. Dure, L. S., Cormier, M. J. 1964. d. Biol. Chem. 239:2351-59 DeLuca, M., Brand, L. 1973. Biochemistry 12:1852 57 138. Bellisario, R., Cormier, M. J. 1971. 128. Henry, J. P., Isambert, M. F., MichelBiochem. Biophys. Res. Commun.43: 800--5 son, A. M. 1970. Biochim. Biophys. A~ta 205:437-50 139. Bellisario, R.. Spencer, T. E., Cormier, M. J. 1972. Biochemistry 11:2256-66 129. Michelson, A. M., Isambert, M. F. 1973. " Biochimie 55 : 619-34 140. Dure, L. S., Cormier, M. J. 1963. J. 130. Michelson, A. M. 1974. Abstr. Am. Soc. Biol. Chem.238 : 790-93 Photobiol. Meet. Vancouver, 62-63 141. Bellisario, R. 1971. Thesis. Univ. of Georgia, Athens, Ga. 131. Michelson, A. M. 1973. Biochimie 55: 465-79 142. Cline, T. W., Hastings, J. W. 1974. 132. Michelson, A. M. 1973. Biochimie 55: J. Biol. Chem. 249:4668-69 925-42 143. Casola, L., Brumby,P. E., Massey, V. 133. Henry, J. P., Isambert, M. F., Michel1966. J. Biol. Chem. 241:497284

Bioluminescence: recent advances.

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