Porphyrin chromophore inLuminodesmus photoprotein

0305-0491/84 $3.00 + 0.00 © 1984 Pergamon Press Ltd

~, pp. 565 567, 1984

C H R O M O P H O R E IN Li PHOTOPROTEIN OSAMU SHIMOMUP.A Laboratory, Woods Hole, MA 02543, U

ESMUS

548-3705)

(Received 27 March 1984) pede Luminoa Abstraet--l. The bioluminescence of the mountain millipeq photoprotein is oxidized in the presence of ATP and Mg.2+, 2 2. The purified photoprotein contained a porphyrin chromophorq cl porphyrin that is directly involved in bioluminescence reaction, reacti making pigments that are present in certain bioluminescent marin~ marme organism~, 3. Based on various circumstantial evidence including g the, the comparison bioluminescent reactions, it is suggested that the light-emitting light-err chro nescence is composed of a partial structure of the porphyrin porphyJ existing

INTRODUCTION he millipede Lurninodesrnus sequoia is one o f the F~rq Lre examples o f bioluminescent organisms t h a t in-

h atbit b i t high m o u n t a i n s of 1500-2000 m altitude in the s o~uthern u t h e r n p a r t o f Sierra N e v a d a (Loomis a n d Davenport, 1951). Greenish-blue light is emitted continauously from the whole body of the millipede by a m e c h a n i s m which involves the oxidation of a p h o t o protein :otein with molecular oxygen in the presence o f A T P a n d M g 2÷ (Hastings a n d D a v e n p o r t , 1957; S h i m o m u r a , 1981). Despite similarity to the firefly minescence in the i n v o l v e m e n t o f A T P a n d M g 2÷ lumines . . . . . . . . ' the Luminodesmus luminescence ;cence does not involve firefly luciferin. The experimental imental results reported here indicate t h a t the photol:protein of Luminodesmus c o n t a i n s a p o r p h y r i n c h r o m o p h o r e . In the bioluminescence reaction, the p o r p h y r i n is p r o b a b l y -type light-emitting chroconverted to a bile pigment-type m o p h o r e which is similar to the th~ bile pigments t h a t are ~uphausiid shrimps a n d present in the l u m i n o u s eu dinoflagellates ( S h i m o m u r a , 1980; D u n l a p et al., 1981). The millipede is strongly fluorescel luorescent scent and, therefore, can be easily located a n d cau aught in the d a r k by the use of a p o r t a b l e ultraviolet light source. The ease of

takes place when a first example of a contrast to the bile mdwidth with other uminodesmus lumirotein.

colleq collection, co~ the distribution of the millipede in a narrow, limited ,~d regio :ion, can lead to the millil enda e n d a n g e r i n g a n d exterminating o f the t~ species if necescaution is n o t taken. Therefor ?herefore, the a m o u n t of sary ~eclmens available in this research has been limited. speci MATERIALS AND METH METHODS The collected in April of 1982 The specimens ( ~ 1500) were collecte alifornia. The photoprotein and 11983 in Camp Nelson, California previously described (Shimwas eextracted and purified as previous omura, 1981) except for an additional cl chromatography step omur (Pharrr that was inwith Phenyl-Sepharose CL-4B (Pharmacia) cluded following the Sephadex G-100 st (Step 3, Table 1). G-100 step Thus, (NH4)2SO 4precipitate from Step p 3 (product from 240 specimens) was dissolved in 50 ml of 10 lC mM sodium phosphate buffer (pH 6.5) containing 5 mM ml~ EGTA and 0.5 M (NH4)2SO4, then added to a column of e Phenyl-Sepharose buffer and the column (2.5 x 10cm) prepared with the same bu Photoprotein was washed with 30 ml of the same buffer. b adsorbed on the column was eluted with 10mM sodium phosphate buffer (pH 6.5) containing 5 mM EGTA and 0.2 M NaCl. SDS-polyacrylamide gel electrophoresis el of the photoprotein was carried out accord according to Weber and Osborn (1969). To split chromophore from the photoprotein, pho 1.3 ml of photoprotein solution was shaken with witl 0.7 ml of ethanol

Tabk 31e I. Purification of Luminodesmus photoprotein from 480 specimensa Recovery of Total Specific activity (A)b A c t i v i t y proteind activity Step (%) saved (B)c (mg) (A/B) 1. Initial extract 8500 69 5900 2. (NH4)2SOb4 precipitate 3. Sephedex G-100 67 3400 148 23 4. Phenyl-Sel: pharose CL-4B 64 1850 37 50 5. Ultrogel AcA 44 68 1080 16 67.5 6. DEAE-Sel:phadex A-50 67 620 8.2 75.6 7. Ultrogel AcA 44 77 410 6.2 66.1 Representative ive data from 3 sets of purification. Starting from 8 batches of 60 specimens each, 4 batches were t following Step 5. ~Total luminescent acti COne unit is equal to li~ measured with a calibrated p

OSAMU SHIMOMURA

:1, then extracted twice pproximately 25% of red protein. The ethyl ashed with 0.2ml of issolved in methanol, e with CrO, and the ~ts by thin-layer chrowere carried out as 80). SSION •,,~o,~L,o ~,,~, ~,,o,_,~oo,v,, As apparent from the data shown in Table 1, the 3cess of purification could not overcome the fast, 3ntaneous inactivation of this unstable photo)tein; the sp. act. reached a maximum at Step 6, t further purification by successive column chrottography after this step caused a steady and :lless decrease in the sp. act. The product of Step which was used in all experiments discussed below, s nearly completely homogeneous with regard to purity of protein according to the results of SDSlyacrylamide gel electrophoresis. Thus, it is consid:d that the active photoprotein and its inactivated "m are inseparable by the procedure currently Lployed. The mol. wt of the photoprotein was determined to D e 104,000 by SDS-acrylamide gel electrophoresis, in contrast atrast to a value of 60,000 previously obtained by gel filtration (Shimomura, 1981). The half-life of the actlvlb :ivity of purified photoprotein in 1 0 m M sodium osphate buffer (pH 6.5) containing 5 m M E G T A phosl andd 0.2 M NaCl was 16 hr at 0 ' C and 45 minutes at 23°C. A & small absorption peak at 410 nm (Fig. 1), as well a s very weak peaks at about 497, 550 and 587 nm ich are not apparent in the figure, must be due to which

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ving activity of 2.51 x 10 ]~ Luminodesmus photoprotein havin quanta/sec/ml (A); same as A with 8 times expansion of ,,ctrum of isolated chromoabsorbance (B); absorption spec phore in methanol, in molar conc{ mcentration roughly equivalent to A, with 8 times expansion m of absorbance (C); in vivo bioluminescence spectrum of a~t CHCl~-anaesthetized live specimen (D); in vitro bioluminescenc ainescence spectrum when en~ lumit ]uminesced in 30ram Luminodesmus photoprotein was Tris-HC1 buffer (pH 7.5) containing 0. 50 # M ATP (E). A, B and C were meas Model UV-240 spectrophotometer, an Perkin-Elmer Model MPF-44B fluores

the chromophc were consistent separate series because the pe proportional tc the weak absor

otoprotein because the) 11 final products of three on experiment and also 410nnl appeared to be ;cence activity, although peak and the instability at allow a ver\ precise of the photop] comparison. T1 ein was non-fluorescent (except u s u a l ] escence) even after the bioluminescent taken place. Th~ chromor ] from the photoprotem by acid a( treatm an absorption peak at 398 nm n: in metl" • The chromophore was practically nonpracti ] neutral solutions, simitein. Acidification of the lar to the origir neutral HC1 acidity resulted in neum solutio] ncrease of the 398 nm sharp•pening an( absorption pea led by a significant inabsor crease in fluore creas( rescence emission peaks 50nm when excited at were found at .'ak was found at 400 nm 400 nm, n] and an when measured and 650 nm in aqueous re was unstable and the 0.1 M HCI. Thl ts quickly lost m 0.1 M 398 nm m absorpl N a O lH wherea y slow change was ohserved in 0.1 M HCI. Dithionite did d not affect the serve( c did 398 nm m peak, suggesting that the chromophore not contain Fe 3+. The chromophore with Th~ oxidation of the isolated ch CrO~ in 2 M H2SO 4 yielded 3-methyl-4-vinylHowever, the oxmaleimide and hematinic acid. H maleil idation idatio at pH 1.2 did not yield the cc~o m p o u n d of mol. 77 that is characteristic of the pyrochlorophyll wt 17 structure (cf. Shimomura, 1980: Dunlap et al.. 1981). that the Thqe results described above suggest s c h r o m o p h o r e of the k u m i n o d e s m u s photoprotein is a chlorophyll-type, in conporphyrin which is not pyrochlorop shrimps trast with the bile pigments of the euphausiid eu and dinoflagellates which derived flom pyro)80; Dunlap el al., 1981 ). chlorophylls (Shimomura, 1980: Du Moreover, because the porphyrin is the only chromoabsorbs light phore in this luminescence system that t ms most likely that in the region above 300 nm, it seem roses from this porthe light-emitting chromophore aris reacphyrin. Thus, in the course ofE bioluminescence biolu robably opened to a bile tion, the porphyrin ring is probablTy ransiently turned into a pigment-type structure or transien structure having conjugation of 2 oor 3 pyrrole rings, which acts as the light-emitter of bioluminescence with an emission maximum of 496 r i m . {ment-type chromoThe formation of such bile pigm phore is supported by the narrow bioluminescence spectrum of this photoprotein. The full width of half )ectrum was only 38 nm maximum ( F W H M ) of the spectrur (Fig. 1, curve E), which is close to the tt F W H M values pigment-type lightof two known examples of bile pi "'F" of emitters: 40 nm, of the fluorescent compound c euphausiid shrimp Meganyctiphane ?hanes, a bile pigment (Shimomura and Johnson, 1968; St;himomura, 1980), and 37 nm, of dinoflagellate luciferi luciferin, a bile pigment . . . . . . . . . . . ) u n l a p el al.. 1981 }. The :hernical types of lighte all considerably larger rot-type: 77 rim, of lumiMNH2 (Hastings et al..

Porphyrin in Luminodesmus photoprot~ Cypridina, involving et al., 1969); 65 nm, le derivative (McE1of aequorin (Shimrn and 70 nm, of the

mp Oplophorus rene (Matthews et al., 50 nm, of Aequorea nvolving a benMorise et al., 1974; himomura, 1979). Structural comparison of the ;ht-emitters suggests that the narrow emission band 'bile pigment-type light-emitters is probably due to Le presence of methene bridges between conjugated ngs in the conjugation system of the chromophore, The apparent quantum yield of the photoprotein as calculated to be 1.0~o based on the mol. wt of )4,000, assuming Att~;n= 20 at 280 nm. The value of ue quantum yield must be much higher than this due because the sample used contained a significant nount of inactivated photoprotein. According to e data of Table 1, approx 90~o was inactivated ~'tween Step 1 and Step 7, and this is in addition to unknown extent of inactivation that occurred Jring the initial extraction. On the other hand, the lta of Fig. 1, curves A and B, indicate that only 5~o oI• the molecules of the protein were bound to the porphyrin )rphyrin group and presumably existed in active form rm when e value of the 410 nm peak (Soret band) is assumed to be a typical value of 2 x 10 ~. If the mple contained 5~o of active form, the true quansam tum yield would become 20%. Acknowl :knowledgement--This work was supported by a research grant ant from the National Science Foundation (PCM8201005).

REFERENCES

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