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Isolation studies with Cypridina luciferin
Isolation
Studies
with
T’ETI
Cypridina
(‘. C’IZ=\IG ANI) 14. S. HAII\‘ET
I”rot,l tllP lalwcl~or~icsof fhe I~odi(y”~lle/~ /nstilttle. I
Luciferin’
sP?0 1-o,+. .I-e,l! I-or/l
September 28, 19GO
,211:rttempt to isolate C’!ypridircn ltic’iferin itI pure form has 11ecr1m:itle. The most 1)romising appronchw have involved I he usr of rollntcrcurrciit distril)Iltion and menof instabilit) t)r:tnc diffusion. Althollgh the fimtl ol)ject,ive W:LS not realized brcn~~sc xnd lack of m:tteri:al, t hc espcrimentr strongly indic:rt,cd the nctivc chromophore to tw :Llow n~olrcw1:rr wiyht slltxst:wce I)ur not of peptitle nature. However, it nppcnretl to kw tightly Ix~nd to peptide mat crixl of unusunl yroperties. At tempts to achieve st:tl)ilia:it ion I)? I)enzoyl:ltiou resultc4 in a mow cornplic:it,cd protluct. Thr use of certain :~nt.ioxidnnt s gave prtial st:it)iliz:ztion.
indicated, something corrtsponding to a molwnlnr weight of 300-500. This was much too low to lw consistent with a chromophoric moiety wpablc of giving the absorption spwtra obwrvcd if it wcrt joined by cwvalent linknpcl to a polypeptidc with the number of ditiercnt amino acids rcportcd in the hydrolyzntcl of the u&w principle isolated by Tsuji, Chase, and Harvey (3). Strangely enough, in the beginning of our own work amino acid analysis of active mat.erial also indicat4 nearly all the common amino a&Is to bc prfstnt. Howver, definite clarification of this paradox was achieved by the finding t.hat. the principle responsible for the lumincscencr could bt separated from the pcptide material bv t.hc mildest of fractionation proccdurrs ’ in which wither st,rong alkali nor arid had bcrn used. In this fractionation plan, the extract, of the C’ypritlina IV:IS subjected to membrane diffusion ill ac~ucw~s solution to remove the inac%ivr molrcwles of largw size. This step could bt> accwmplished with but little loss of luminrscent activity. The rwiduc recovered was then subjected at “5” (~01111tercurrrnt distribu_. t 0 a ?A-stage tion in a system made from glacial acetic ~Lc~id~n-hutanol-?~-hcpt:~lle--mtt hanol (4 :3 : I:1 v v). Soon after our studiw were begun, a paper by .Japuncsc~ workers (5) cam? to our attcntion ill which isolation cxpcriments wrc report cd on considerably larger amount :: of fresh organisms. I:ollowing thv bcnzoylntiou prowdurC of Xndwwl~ (ti), t hr> bcnzoyl protl~wl~ was hydrolyzed and further frac>t ionat cd by partition c.hronl:Ltogl.aphy. ;I few milligrams of crystallilw, highly activt mntvrial MXS thus obtainrd \vhirh later was found by Hirata cl nl. (7) to give only tmws of :tmilw arids on hydrolysis. Suficirnt~ niatvrial was obtaintd for ultimate analysi,G and thr dcrivat~ion of a11 empirical formttln. Their st udirs obviously have progrcswd f~lrthor thall otws unless their lwl~zoylation prowdur(~ had considcrnbly :altrrrd t,h(, molwulv in spitv of its retcwtioil of luminrwrnt activity. Olir rrsults as arc not i,wonsistc~l~t with the .Japanrw work. WC also prrparrd thf bc~~zoyl product and stridivd it vxtc~llsi\Y~ly by (‘o~liltc’rcurrrllt disfiL1.
:ls
thc>y
go
tribution. This work, contrary to our cxpericncr with t,he unmodified material, seemed to indicut8e a multiplicit,y of active principles or possibly modifications resulting from the t,reatmrnt. It was furt,her found t,hat, the benzoylnted product., ahhough considerably morr stable than t,he free matrrial, also slowly underwent, decay whrn exposed to oxygen. A thorough search was madr for othrr ways of st.abilizing t,he act,ivr principle, by addition of ant,ioxidant,s to t,hr system 114 for c,o\lntcrcurrent, dist,ribut,ion. Ascorbic acid and sodium hydrosulfite srrmrd to have a rrtarding effect, hut, mrrr not coniplrtely rffwtivr as stabilizers. ISl’ERIMEiYTAI.
ASD
RESULTS
P!~pritlinn organisms, collected between l!Mi1952 in the sea of Japan and dried in the sun, were parkagetl for storage in sealed bott.les containing bags with CnCls Ext,rnct.ion experiments were done rit.her with whole organisms or with pow dered material. In some esperiment.s the latter was defatt,ed by benzene and ether estract)ion in a So-uhlet, apparatus (2-3y0 of the dry weight).
h quantitative est,imat,ion of luciferin activit) during all operations was done with a MacKichol l)hotomult,iplier photometer (8)) employing the enzymir reaction with lllciferase in 0.067 M (M/15) phosphate buffer, pH 6.8, made 0.1 .Y in SaCI. Recordings were made with an Esterline-Angels recorder. A special chaml)er in front, of the photomultiplier window was built* to wcommotlata R test tube where thr components of the luminescent reaction were mixed. Luciferin solution was introducrd first (usually 0.1-l .O ml. depending on the conrentrnt,ion), and after t,he light meter was set for recording of either instantaneous or integrated light. :I measured quantity of enxymc solution (0.50 ml. iI1 10 ml. M/15 phosph:tt.c buffer. plI 6.8) was uddrd by means of n syringe equipped with :t long stainless steel needle. containing it built-in valve, and extending to the bott,om of the test tulw. The errzymc solution was ntlded through an opening in a. rlkbber stopper to insure complete darkness in thr mixing chamber. The light. uuiis * \Ve are indebted Rockefrllrr Institute
to ;\lr. 1Verner for his help.
Ii. Iirl1g
of the
CTPRIDISA
(1.11.) used in this paper are arljitrary and correspond t,o volts on the voltmeter scale of the recording paper.
Extraction f’roretlroe 21. A 50-g. sample of dr,v Cypridina organisms was placed in a column provided with a glass-frittetl disk and attached to a suit:tl)le flask immersed in an ice-water bath to permit collection of s:~mplcs rlntler partial vacuum. (ilass wool was placed on the top of the column to prevent floating of the matjcrial and the extraction was carried otlt with boiling water appl.ving a gentle suction. Fifteen samples (28 ml./sample) were collcct,etl, and the artivit)y was dct~crmined with luciferaw. Under these conditions the luciferasc originally present in the organisms is tlcnaturrd with hot, wat,er t,hus enabling extraction of native lucifcrin. The latter was found predominantly in the first seven tulles, and the total yield of activity from the wnple n-us 3608 1.u. (0.74 I.u./mg. of the original weight of C'ypridirm). I’rocctl~rr: W. 1)irert extraction of the whole organism (100 g.) with boiling methanol (500 ml.) gavr 7tioo 1.u. I'rwrrlrcrc ('. Finely ground Cypridina previowly def:l~ttetl with benzene was extracted at 4” rmbr nitrogen in a column :is in Procedure A with methanol wntaining 0.17~; ascorbic acid. The total activit)- rccovercd in the first fractions \vas similar to l’rowdurw A and IS.
Protection of Lucifuin I~atrs of autoridation of methanolic luciferin sollltions were determined under several conditions. Unprotected solrltion stored at 4°C. in :I glass-stoppered centrifuge tube lost half of its activity aft.rr 1 t1a.v. Storage at -20°C. or in 0.154 mcthnnolic solution of ascorbic acid at 4°C. prolongr~l the time for 5O;c O* loss of artivit\. to 12 days. In some countercurrent distribution csperimrnts (see below), a O.l~;~, solution of NazSzOi was cased for a pa&t1 protection of activity. This salt had I)een shown (9) to be capable of reversing autoxidation of luciferin to ‘iO(i, provided the reduc*tion was carried out immediatelv aft,er osidntion.
Solwnt Fractionation dlethorl I. The methanolic residue of crude lucifrrin obtained from a 50-g. sample of Cypridina powder (Procedure C) w-as extracted at room temperature with three 50.ml. portions of isopropyl alcohol in a partially evacuated flask. The combinrd extracts were cvaporatcd to 3 residue, and the lai ter was extracted with three 50-ml.
LUCIFERIS
30::
portions of chloroform. The chloroform-soluble residue was only partially soluble in water, Ijut most of t,hc activity(25-50’:; of the original) \\-a~ cstracted wit,11 three 50-ml. portions of water. Method ZZ. In this procedure a methanolic estract, (l’roccdurc C) bvas concentrated to a small volume follo\ved 1)~ the addition of a tenfold excess of ice-cold 0.5 .V HCI. After removal of the insolul)le material eit)her 1)~- filtration or centrifngation, the clear solution was est,racted with ether until the extracts became rolorlcss followed 1)~ an extraction with n-butanol. The partition constant jKL) determined l)y light measurement on rqrd volumes of the two phases \V:IS approsimately 20 in favor of n-butanol. A qu:tntit,ative experiment with a sample containing 134 1.~. in 50 ml. of 0.5 .\’ IICl indicated t,hat two est,ractions at 4°C. with qua1 volumes of n-tmtanol were sufficient to rcmove lucifrrin from the aqueous phase and indicated a &fold concentration of activity. Method ZZZ. In anothrr experiment 80 g. C',ypritlinn were extracted as in Procedure A. The cooled aqueous solution (200 ml.) was extracted with nI)utanol (200 ml.) at, 5°C. >-ielding 5000 1.11. The organic layer was extracted with two 200-1111. partions of cold water whirh removed approximnt~r~ly 10% of activity together with water-soluble inactive material. Subsrquentl,v the organic ph:we (170 ml.) was diluted with 1 vol. ?a-heptanc and extracted three times with 200-ml. portions of cold 0.1 S acetic wid. This served to bring lllc*iferin into the aqueous phase leaving behind waterinsoluble impurities in the organic phase. The romhined aqueous est.r:tct,s (808 1.11.) were extracted with 100 ml. wl>utanol ,vielding a residue with 750 l.u. (15C’, 20 l.u./mg.). Large losses were due to the oxidation of luciferin, resulting in part to the formation of stable emulsions which prw longed the exposure to air. Ncvert helcss, this procedure yielded, without the use of a mineral acid, the best preparat,ion of crude luciferin rcl:itively free from large quant it,ies of polar and now polar impurities.
Early dialysis experiments with luciferin preparation ohtuined by solvent fractionation, Method I, in water ml&r nitrogen nt’rnosphere with two types of acllophalle nmnhranes (Visking cellophane dialysis tubing 4-f:s2 and 23 / 32) established the escape rat,es with half-times (t/z, time required for the escape of 50 5; material) l,et~v-rcn 3:3 and 36 min. The esperiments were patterlied aft’cr the work of Craig and Icing (lo). Dialysis was followed hp weight, optical
density, and luminescence mcasurement,s. On the basis of t,he comparison of the rat,c curves obtained wit’h t’hose of other known compounds obtained under similar conditions, it’ was inferred that the molcculal weight) of luciferin is Mweeu 300 and 500. In a later experiment, a sample (70 1.u.) of the luciferin prcparat.ion obt,ained by solvent fractionation, Method II, was dissolved in 0.5 ml. of 0.1 %’ ;“\;a&Q solution in water (pH 4.5), centrifuged, and the supernatant was dialyzed at 20°C:. in an analytical cell wit,h acetylatcd 1852 Visking cellophane tubing against, 10 ml. of the same solvent. The dialysis was performed according to the met,hod of Craig, King, and St,racher (I). The half-t,imc of escape (t/2) t,hrough the membrane was 230 min. w&h a total rcco\:ery of 10.73 1.11.(14.8 ‘I;). With an unmodified I$/32 membrane a similar sample (70 1.11.) in 0.1 ‘i; XaBz04 under the same conditions gave a t;‘2 of 125 min. with a recovery of 66.46 1.11.(95 %). Subt’ilin (4.5 mg., molecular weight, 3200, 0.8 absorbnncy units,‘mg.,’ ml.) was taken as a reference compound giving in the same solvent with 1552 membrane t/2 366 min. with a recovery of 0.34 absorbance units at 280 rnp. In tbe second series of experiments, 0.01 N acetic acid was used as a dialyzing medium. With a sample (38 1.1~) dialyzed through 1932 membrane at, 23”C., t,/2 was 103 min. and t,he recovery 37.7 1.1~ ((3070. Under similar conditions subtilin (5.8 mg.) TABLE RATES (tlS DIFFERENT
Ihr,us~s
min.)
‘9*,2
membrane Substance 0.01 s acetic acid
Luciferin Subtilin Luciferin
+
pa
p:tin HCl-treated ferin Itedialyzed ferin
luciluci-
I
OF LUCIFERIN COXDITIONS
UXDER
acetylated ‘562 membrane
had a t,!2 of 80 min. with the recovery of 0.834 absorbance units at, 280 mp. Wit,h an acc%ylated 1q2 membrane and 0.01 N acetic acid, a lucifcrin sample (60 1.11.)gave t ‘2 400 min. at 22.5”c1. with t,he recovery of 50 1.11.(83 ‘X). It, is evident from Table I t,hat for the same preparat’ion of luciferin t,he nat’ure of the membrane and the dialyzing medium have a pronounced effect on the magnit,ude of t/ 2. httfmpts were made to hydrolyze this luciferin preparation with papain and t,o dialyze the resulting reaction mixture to see whether t)herc was a change in the escape rate due to the treatment’. A sample (62.4 1.11.)was dissolved in 1 ml. of 0.01 ‘;. cystcine solution in 0.067 A1 phosphat,e buffer (pH 6.8), reacted overnight’ at room temperat,ure with 4.2 mg. of two-t,imes rrystallizcd Worthington papain prcparat)ion, and dialyzed in 0.01 N acetic acid with 18,iZ membrane giving t/2 160 min. with t)he recovery of 21 1.~. (34 ?i ) (Table I). The significant retardation of diffusion rat,e could he due to the association of luciferin complex wit)h the rnzymc.
It was of int,erest to compare the diffusion rate of luriferin treated with 0.5 Ar oxygenfree hydrochloric* arid at) 100°C. for 1 hr. with that of untreated mat,crial. This is the hydrolysis method used by Anderson (6) for t,he drbenzoylation of benzoylluciferin and by Hirata d al. (7) for the preparat,ion of the crystalline lucifrrin. X sample (84 1.u.) subjected to such a hydrolysis with 3.5 ml. of 0.5 S HCl and 0.5 ml. n-but)anol in an evacuated tube and recovered in the form of a residue was dialyzed at, 24°C:. with an l:$z membrane in 0.01 N acetic acid. This gave a t/2 of 25 min. and with t)hc recovery of 71 1.11.(85 96). ITrider t,hcse conditions of acid treatment,, peptide bonds rnight be hydrolyxcd and give rise to smaller, but, still active, fragments of luciferin. To avoid t,his possihilit’y, :I fresh sample (2,500 1.11.)of luciferin cxt~rnct was dissolved in 10 ml. of 10 R) methanol in 0.1 N arctic acid, placed in ly$z tubing and dialyzed at. 5°C. mldrr pure nit,rogen against, five loo-ml. volumes of 0.1 A’ acetic acid. After this no activity was found in the dialyxatc. I’ndrr t hew conditions the t/2
was :jOOmin. with t,he cluantitativc re~o~ry of activit,y. The dialyzntes were combined, nlld usrd for corltitc,r.currcilt w:tporatrd, distribution as dcscrihcd below. A sample (20 1.11.)of this material was dinlyzcld in an analyt~ical cell (4) with an l,@$z membrane in 0.01 S acctia acid giving a tL? of 10 min. with the rcco\-cry of 17 1.11.(85 ‘Y). The first, dialyxatc (10 ml., G.8 1.u.) was used t,o study thr spontanrorw loss of activity during the timcl of dialysis (64 min.). It was rcdaccd to 6.2 1.~. (!)I %). The result of this cxpcrimcnt indicates that the first dialysis NYLSsufficient to rcmovc a cwnplcxing irnpurity rcsponsiblc for the initial slow diffusion rate of lrwift~rin.
activity of ~65 l.u.,/mg. indicatjing a purification of 6000-fold over the dried organisms. Thr cut shown (tulws 8-23) \vas takrn fol rccowry. The absorptiou spectrum of this mntcXrial ill mct~hanol shown in I;ig. 1-,was similar to that rrportcd in thr litwaturc (3, 5), hut’ obviously partial oxidation had alwad> taken pl:wc. Amino acid analysis by t hc m&hod of 1Ioore, Sparkman, and Skin (I I ) showed insignificant, amounts of arnillo :wid twcpt lysine, wpartic ac’id, glutamir wid. and isolrucine. (‘.CJj.
with
Ed-act
from
diethod
/I1
=\fter investigating sevcwl systems, tht> most promising one wit,h thcl rcsidur from .1fter preliminary trials the extract residue this txtract swmad to he WC made from :I solution of 0.1 ‘:I sodium hydrosulfitc~ (‘onfrom 200 g. of the dried organisms which acitl~n-hlttarlol--ngave 12.000 l.u. was distrihutcd to 100 taining 0.1 4 astir trausfcrs’ at 4” under purr nitrogen in the h~ptanc-mc~thallol (1.8:0.75: 1.%5:0.2 v v). system 0.01 S HCll (containing 0.01 5% Hccauw of tht> knowrl stabilizing cffcc*t of ascorbic a~id)-~y~lohexarl~~?~-l~~lt:li~[)l (2 : YazS.J4, an atkmpt was made to carry out I : I v/v). The phase \-olumrs wcrc :L$ ml. the distribution at 25” in a C.C.D. apparut.us with 1000 tubes (a,?? ml. phase volrmws;). .I Thr~ weight (nftcr correcting for aworbic acid) and activity patterns (in terms of run of 1000 transfers rcquircd se\-cral (1:~~s volts; ml.) :MY shown in Fig. la. The most, of cwntinuous operation. The rcsidlw sample wtivc material was that from tubs SO-70 containrd 2800 1.~. alld weighed ahout 1-M which npprowhcd 59 l.u.,/mg. or about w. 9X-fold purifkat ion from the dried organAnalysis was made of both rlppw and lower phases try luminescent, activity and isms. ,&Jut half the total light units origillall>’ talwrr could h awountcd for in the hy optical density at 4% rnl.c. Only two pat tern. regions of activity n-we found lvhicah COYTn~o cuts were takrn as shown in l’ig. la rrspondrd to thr opt,ical density hands for rwovwy. The rrsiduc from cut, 2 (15.8 shown in F‘ig. 3. However, t.he hands at 1.11.‘mg.) was redistributed under nitrogrn t,ubw 325-400 had much higher acativit y at 1” to 100 transfers in the system 0.01 N than the hand at, 50-I 00. The total rrcwvwy HC’l-ethyl :Icrt:ttc-n-~)utanoI-mcthallol (3: of light was poor, ahout 563 l.u. or 20 “; of :I:O.25:0.5). This gave the pattern shown in thr starting sample l+?g. lb. The first band had less specific The material in the most, active t)and, acti\-ity (20 1.11.‘mg.) than the broader t nhes 535420, was rcwwwd by rl-aporation scc~~ntl hand (!I I l.u.,t mg.) which uworurtjcd in a rotatory rvaporator, taken up in 50 nil. for 75 “; of thr activity. Howwr, in all only n-butanol and rxtracted t)hrec timrs wit,h 30 ahout one third of thr total activity was ro- ml. watrr t,o rcmow inorganic: salts. The co\-cwd. organicfi layrr gave i.2 mg. rrsidw which .I ccwtclr cut (tubes 30-50) from the I\-idcr still had :L sprcific activity approximating halltl VW rwo\wcd and rcdistributcld ill the Cl 1.u.’ rng. and showrd thr typical ahsorpSNllf’ s.vstcm rmdcr the snmc conditions. t ion specl ruin of partly oxidizrd IliciferiIi This gnvc pattwll lc. Only one active lland (3). was now olwrwd with a K approximatirlg Part of this material was hydrolyzed in (j that of t ht> first hand in the pwwding pat2V IICl and analyzed for nrnillo acids by the tcrll. ‘I’hc> most wtivc frac+on had a spwific nwthotl of Aloow, Spwkman, and Stciin (I I.). (‘.(‘./I.
with. E&act
from
AI&hod
II
12 2 10
9 P ” 0.0
FJ$ .c x 6 .‘j: ; 2 4r 5 : 2 .-2 E
0.6
i
2 0
20
40
60 Tube
80
20
100
40
60
Tube
No.
60
No 6)
(a)
365l.u./mq. I
HI
I
Iy,LvL^hl
;
100 40 60 80 Tube No. Cc) FIG. 1. (a) C.C.D. patterns with an extract from Method II; (h) pat,terns from redistribution l.u./mg. matjerk from (a); (c) patterns from redistribution of !)1.2 l.u./mg. material from (h). 20
with Extract from Method Membrane Di$usion
of 58
The t#ot,alrecovery of amino acids accounted for only about, 23.57~ of t)he sample. All the
C.C.D.
common
Since the material active with luciferast after passing t,hrough t,he membrane had different properties, a distrihlrt8ion was at-
amino
acids
except
were present in ratios covering not more t,han 1 to 5.
tryptophan 51 spread of
iII
after
CTPRIDIKA
Wave length FIG. 2. Absorption
spectrum
curve
307
LUCIFERIS
of material
- rnp recovered
from
distril-xitil
r
n shown
in lc
I
-1
- 2.4
0.6 I 0.5 $
0.4
3 2 Fi d
0.3 0.2 0.1
100
200
Fra. :S. C.C.11. pattern
300
400
500 600 700 800 Tube No. from 1000-transfer distribution of extract
900
1000
from i\Iethod
TIT.
308 z
0.40
T 2 + 0.30 ii Ek 2 0.20 $ 3 0.10 t’ a Fi d 40 FIG. 4. C.C.1).
80
120
200 160 Tube No.
pstt2ern of material
alt,hough it was considerably more narrow t,han a calculated curve. Recovery of this band gave a few milligrams of residue with t,he typical absorpt,ion spect,rum of partly oxidized luciferin. After hydrolysis, amino acid analysis by the ionexchange met,hod of Moore, Spa&man, and St’ein (11) show-cd only traces of serine, isoleucine, and lysinr: tot,aling not more than 2 7%of t.he residue hydrolyzed. EXPEHIMEXTS
WITH BETYZOYL~TEI) I,r-ClFERIiY
A mcthanolic extract, of defatted (“yppowder was evaporated to a residue and the residue suspended in n-hutanol. After centrifuging off t(he insolttblc nMeria1, the solutjion was used dircct,ly for henzoylnt,inn hy the method of iZnderson (6). For example, a Xl-nil. solution containing approximately 6000 1.11. in n-but,anol was renct.ed at 0” tmdcr vigorous shaking with t.hree successive 1-ml. port.ious of benzoyl chloride added dnriug 1 hr. The solute was now found not to he able to luminesce in the st,andard test with Iucifcrasr. After standing an additional hour, the solution was treated tvith 50 ml. water and ext,ract,cd with rt.her. The ether cxt.ruet, was concent’rated to aboutj %5 ml. and diluted w%h prtroleum ether until no activity (following hydrolysis as given below) remained itI the solution. The active precipitate was then taken up in ethyl ether and ext’ract’ed \vit,h 5 ‘/ XaHC’Oa followed by 10 % acet,ic
ridiaa
from hlet.hod
240 III
after
280
320
dial,wis.
acid. The henzoylatrd material was ucutral and remained in the ether. The oily residue obtained on evaporation of t,he ether could be reconverted t.o a sttbstance which gave light’ with luciferase hy treating it with 0.5 :V HCl and n-hutanol in a sealed evacuated t,uhe for I hr. at 95100”. Before evacuation the oxygen in the tutu was replaced by pure nit,rogen. The rwovcry of act,ivity seemed to range from 25 to 50 ‘T; of t,he original. ;\lemhrane diffusion (-2) experiments were made on t,hc hrnxoylnt~ed residue. In contrast to the active unhrnzoylated extract, tho fwnxoylatcd material was not soluble in water. The diffusion cxpcrinw1t.s were, therefore, made irr 7.5 5: aqueous methanol at room temperature. With 19;;~ Visking most of the activity (measured after hydrolysis) escaped with a t/2 of -20-55 min. Even after 18 hr. ahout 10% of the herlzoylated activity did not diffuse through the membrane. 1Vit.h the more porous 2352 Visking, however, all the hcnzoylated activity readily diffused out. The ryi2 membrane, therefort, gave definite fractionation. A large sample of the benzoylatcd prcparat,ion was frwtionated by dialysis through lY&( Visking and subjected t,ct C.C.D. in a system made from nlethallol~c’y”lohexane-ethyl accbtc-water (2.2 : 2 : A : 1 1,/v). In a 1000 tube 2,‘2 ml. machine, l13A transfers were applied. The pattern oht,ained is shown in Fig. 5. At intervals across the pattern material was recovered, hydrolyzed a,nd
2.0
1134 T
0.29 (
-I
0.40
1.6 v--4 F 1.2 N 2 R 0.8
0.12
0.06 t
0
0.4
0
100
zoo
300
400 500 600 Tube No.
‘700
800
900
ii8 0.20 gCL 0.10 11000 0.30
310
MARFEP,
CRAIG
Indeed, hydrolysis may have occurred hut the smaller molecular size of the acti\-it,> was clearly indicated by recovery of act,ive diffusate through I,8 iy Visking cellophane in 0.01 :\: acetic acid mit’hout. prior hydrolysis and thcll rcillvest,igat,ion of it,s diffusion rat,c. As can be seen from Table I, diffusion now was even mow rapid t,han with the HCltreated preparation. The effect. of the membrane diffusion in removing luciferin from the material to which it was hound was further shown by the difference of the original cxt,ract and the diffusatc in countercurrent distribution studies. Moreover, the smaller size of t!he luciferin was also shown by mctnhranc diffusion studies wi-ith the twnzoylat,ed lucifcrin. Here, hecause the benzoylat,cd derivative was not soluble ill wat,er, i5%, methanol was used. This is known (10) to ret,ard greatly the dialysis rat,c of various solut#es. &uit)e apart from the membrane diffusion studies the c~ounttrcurrcnt distribution studies indicated a strong associat,ion of the activr principle t’o inactive material. This was shown by the fact that, a K determination in a suitable system on the basis of luminescent activity with a crude extract, would show a considerable shift in K with hut’ a few transfers. The succrssivc distributions shown in Fig. 1 with different systems indicated a major problem to he that of disengaging thr active principle from the peptide material to which it was bound. Figure 3 is equally ii&resting in this connection. Herr an attempt was made to make a prolonged distribution at room temperature in the prcscnce of t,he known stabilizer (9), sodium hydrosulfite. After 1000 transfers the majority of t,he remaining activity was in a single band which seemed to behave almost ideally frotn t,hr standpoint of countercurrent distribution. However, the material in t,his band did not have the specific activity expected, and amino acid analysis after hydrolysis indicated nearly at1 the known amino acids to tw present. Evtn to distribute in this way in such a nonpolar system would rccglirc that t,hese amino acids arc combined to form a molecule wit’h no effective polar groups. Evtn t,hc cyclic an& biotic polypcptidcs devoid of basic or acidic
AND
HARVEY
groups have shown more polarity than these substances. The experience here is nnlch like the chromatographic expericncr of Tsuji ct al. in attempting to isolate C’ypridina luciferin in pure form. Chromatographic: experiments in this laborat,ory also gave rcsult,s sitnilat to theirs. Apparently the conditions used did not have suflicient dissociating st,rengt,h t,o permit separation of the active principle from the accompanying peptide material. When similar tnatcrial was studied except that it, had dialyzed t’hrough 1yi2 Visking cellophane, the system used in l:ig. 3 was no longer suit~ablc. A more polar system was required, and the result shown in E’ig. 4 was obtained after ?,-I4 t,ransfcrs. The single act,ive band was not ninhydrin positive, and after hydrolysis amino acid analysis showed them to be almost cntirrly absent. This experiment, in spite of ot,her experiments to the cont,rary suggests that the active principle in the gland is a relatively small solute which can tw dissociated and separated from pept’idc mnt,crial of very unusual character. The active principle still can tw of cyclic nature derived from amino acids. While all our cxperitncnts with free Cypridina luciferin were ronsist,ent with this view, those wit’h t,he henzoylat)ed mat’crial were not. The t)enxoylat,ed material distributed as a very c*omplcx mixture as pattern in Icig. 5 shows. The partition ratios across the chart, (comparison of upper and lower optical drnsitics) arc consistentJ with t,he tube numtwr in which the solute occurs. Hydrolysis and luminescent asslty indicated activity scattcrcd at1 across the chart,. The numbers ahovc t,he curves give the l.u./‘mg. (lomplctc hydrolysis of the material in sevcral rrgions and paper chromatography indicatcd numerous ninhydrin-positive spots corrwponding to amino acids. The cut of tubes 350-425 was recovered and rcdist,ributcd tmiw to 120 transfers in the system glacial acetic acidPwatcr-cyclehcxanf-met hanol-chloroform (2 : 1 : 1: 1 : 2). This gave a single baud of about theoretical Ividth, but on hydrolysis and lumincsccnt. assay the activity was found to be increased only to 0.82 l.tl./mg. The hcnzoylation experiments taken alone
C!TPl~ll)IS.1
wem to ildi(xt(~ that (‘~ypn’dina lucifcrin is an activr c~hromophorc c~onjugatcd to LI hetcrogcncow spwtnun of pcptide materisl. This seems to Ix supportctl by other fmct,iolintion rxperimcwt,s inchidilig various forms of chromatography. While the henzoylatcd matrrid ww mwh more stahk than the frrc lwifcrin, it also W:LSfound to low slowlp the ability to lumincsw with lucifcri~l following hydrolysis.
LTTCIFEIt
IN
::I 1
Studies
with
T’ETI
Cypridina
(‘. C’IZ=\IG ANI) 14. S. HAII\‘ET
I”rot,l tllP lalwcl~or~icsof fhe I~odi(y”~lle/~ /nstilttle. I
Luciferin’
sP?0 1-o,+. .I-e,l! I-or/l
September 28, 19GO
,211:rttempt to isolate C’!ypridircn ltic’iferin itI pure form has 11ecr1m:itle. The most 1)romising appronchw have involved I he usr of rollntcrcurrciit distril)Iltion and menof instabilit) t)r:tnc diffusion. Althollgh the fimtl ol)ject,ive W:LS not realized brcn~~sc xnd lack of m:tteri:al, t hc espcrimentr strongly indic:rt,cd the nctivc chromophore to tw :Llow n~olrcw1:rr wiyht slltxst:wce I)ur not of peptitle nature. However, it nppcnretl to kw tightly Ix~nd to peptide mat crixl of unusunl yroperties. At tempts to achieve st:tl)ilia:it ion I)? I)enzoyl:ltiou resultc4 in a mow cornplic:it,cd protluct. Thr use of certain :~nt.ioxidnnt s gave prtial st:it)iliz:ztion.
indicated, something corrtsponding to a molwnlnr weight of 300-500. This was much too low to lw consistent with a chromophoric moiety wpablc of giving the absorption spwtra obwrvcd if it wcrt joined by cwvalent linknpcl to a polypeptidc with the number of ditiercnt amino acids rcportcd in the hydrolyzntcl of the u&w principle isolated by Tsuji, Chase, and Harvey (3). Strangely enough, in the beginning of our own work amino acid analysis of active mat.erial also indicat4 nearly all the common amino a&Is to bc prfstnt. Howver, definite clarification of this paradox was achieved by the finding t.hat. the principle responsible for the lumincscencr could bt separated from the pcptide material bv t.hc mildest of fractionation proccdurrs ’ in which wither st,rong alkali nor arid had bcrn used. In this fractionation plan, the extract, of the C’ypritlina IV:IS subjected to membrane diffusion ill ac~ucw~s solution to remove the inac%ivr molrcwles of largw size. This step could bt> accwmplished with but little loss of luminrscent activity. The rwiduc recovered was then subjected at “5” (~01111tercurrrnt distribu_. t 0 a ?A-stage tion in a system made from glacial acetic ~Lc~id~n-hutanol-?~-hcpt:~lle--mtt hanol (4 :3 : I:1 v v). Soon after our studiw were begun, a paper by .Japuncsc~ workers (5) cam? to our attcntion ill which isolation cxpcriments wrc report cd on considerably larger amount :: of fresh organisms. I:ollowing thv bcnzoylntiou prowdurC of Xndwwl~ (ti), t hr> bcnzoyl protl~wl~ was hydrolyzed and further frac>t ionat cd by partition c.hronl:Ltogl.aphy. ;I few milligrams of crystallilw, highly activt mntvrial MXS thus obtainrd \vhirh later was found by Hirata cl nl. (7) to give only tmws of :tmilw arids on hydrolysis. Suficirnt~ niatvrial was obtaintd for ultimate analysi,G and thr dcrivat~ion of a11 empirical formttln. Their st udirs obviously have progrcswd f~lrthor thall otws unless their lwl~zoylation prowdur(~ had considcrnbly :altrrrd t,h(, molwulv in spitv of its retcwtioil of luminrwrnt activity. Olir rrsults as arc not i,wonsistc~l~t with the .Japanrw work. WC also prrparrd thf bc~~zoyl product and stridivd it vxtc~llsi\Y~ly by (‘o~liltc’rcurrrllt disfiL1.
:ls
thc>y
go
tribution. This work, contrary to our cxpericncr with t,he unmodified material, seemed to indicut8e a multiplicit,y of active principles or possibly modifications resulting from the t,reatmrnt. It was furt,her found t,hat, the benzoylnted product., ahhough considerably morr stable than t,he free matrrial, also slowly underwent, decay whrn exposed to oxygen. A thorough search was madr for othrr ways of st.abilizing t,he act,ivr principle, by addition of ant,ioxidant,s to t,hr system 114 for c,o\lntcrcurrent, dist,ribut,ion. Ascorbic acid and sodium hydrosulfite srrmrd to have a rrtarding effect, hut, mrrr not coniplrtely rffwtivr as stabilizers. ISl’ERIMEiYTAI.
ASD
RESULTS
P!~pritlinn organisms, collected between l!Mi1952 in the sea of Japan and dried in the sun, were parkagetl for storage in sealed bott.les containing bags with CnCls Ext,rnct.ion experiments were done rit.her with whole organisms or with pow dered material. In some esperiment.s the latter was defatt,ed by benzene and ether estract)ion in a So-uhlet, apparatus (2-3y0 of the dry weight).
h quantitative est,imat,ion of luciferin activit) during all operations was done with a MacKichol l)hotomult,iplier photometer (8)) employing the enzymir reaction with lllciferase in 0.067 M (M/15) phosphate buffer, pH 6.8, made 0.1 .Y in SaCI. Recordings were made with an Esterline-Angels recorder. A special chaml)er in front, of the photomultiplier window was built* to wcommotlata R test tube where thr components of the luminescent reaction were mixed. Luciferin solution was introducrd first (usually 0.1-l .O ml. depending on the conrentrnt,ion), and after t,he light meter was set for recording of either instantaneous or integrated light. :I measured quantity of enxymc solution (0.50 ml. iI1 10 ml. M/15 phosph:tt.c buffer. plI 6.8) was uddrd by means of n syringe equipped with :t long stainless steel needle. containing it built-in valve, and extending to the bott,om of the test tulw. The errzymc solution was ntlded through an opening in a. rlkbber stopper to insure complete darkness in thr mixing chamber. The light. uuiis * \Ve are indebted Rockefrllrr Institute
to ;\lr. 1Verner for his help.
Ii. Iirl1g
of the
CTPRIDISA
(1.11.) used in this paper are arljitrary and correspond t,o volts on the voltmeter scale of the recording paper.
Extraction f’roretlroe 21. A 50-g. sample of dr,v Cypridina organisms was placed in a column provided with a glass-frittetl disk and attached to a suit:tl)le flask immersed in an ice-water bath to permit collection of s:~mplcs rlntler partial vacuum. (ilass wool was placed on the top of the column to prevent floating of the matjcrial and the extraction was carried otlt with boiling water appl.ving a gentle suction. Fifteen samples (28 ml./sample) were collcct,etl, and the artivit)y was dct~crmined with luciferaw. Under these conditions the luciferasc originally present in the organisms is tlcnaturrd with hot, wat,er t,hus enabling extraction of native lucifcrin. The latter was found predominantly in the first seven tulles, and the total yield of activity from the wnple n-us 3608 1.u. (0.74 I.u./mg. of the original weight of C'ypridirm). I’rocctl~rr: W. 1)irert extraction of the whole organism (100 g.) with boiling methanol (500 ml.) gavr 7tioo 1.u. I'rwrrlrcrc ('. Finely ground Cypridina previowly def:l~ttetl with benzene was extracted at 4” rmbr nitrogen in a column :is in Procedure A with methanol wntaining 0.17~; ascorbic acid. The total activit)- rccovercd in the first fractions \vas similar to l’rowdurw A and IS.
Protection of Lucifuin I~atrs of autoridation of methanolic luciferin sollltions were determined under several conditions. Unprotected solrltion stored at 4°C. in :I glass-stoppered centrifuge tube lost half of its activity aft.rr 1 t1a.v. Storage at -20°C. or in 0.154 mcthnnolic solution of ascorbic acid at 4°C. prolongr~l the time for 5O;c O* loss of artivit\. to 12 days. In some countercurrent distribution csperimrnts (see below), a O.l~;~, solution of NazSzOi was cased for a pa&t1 protection of activity. This salt had I)een shown (9) to be capable of reversing autoxidation of luciferin to ‘iO(i, provided the reduc*tion was carried out immediatelv aft,er osidntion.
Solwnt Fractionation dlethorl I. The methanolic residue of crude lucifrrin obtained from a 50-g. sample of Cypridina powder (Procedure C) w-as extracted at room temperature with three 50.ml. portions of isopropyl alcohol in a partially evacuated flask. The combinrd extracts were cvaporatcd to 3 residue, and the lai ter was extracted with three 50-ml.
LUCIFERIS
30::
portions of chloroform. The chloroform-soluble residue was only partially soluble in water, Ijut most of t,hc activity(25-50’:; of the original) \\-a~ cstracted wit,11 three 50-ml. portions of water. Method ZZ. In this procedure a methanolic estract, (l’roccdurc C) bvas concentrated to a small volume follo\ved 1)~ the addition of a tenfold excess of ice-cold 0.5 .V HCI. After removal of the insolul)le material eit)her 1)~- filtration or centrifngation, the clear solution was est,racted with ether until the extracts became rolorlcss followed 1)~ an extraction with n-butanol. The partition constant jKL) determined l)y light measurement on rqrd volumes of the two phases \V:IS approsimately 20 in favor of n-butanol. A qu:tntit,ative experiment with a sample containing 134 1.~. in 50 ml. of 0.5 .\’ IICl indicated t,hat two est,ractions at 4°C. with qua1 volumes of n-tmtanol were sufficient to rcmove lucifrrin from the aqueous phase and indicated a &fold concentration of activity. Method ZZZ. In anothrr experiment 80 g. C',ypritlinn were extracted as in Procedure A. The cooled aqueous solution (200 ml.) was extracted with nI)utanol (200 ml.) at, 5°C. >-ielding 5000 1.11. The organic layer was extracted with two 200-1111. partions of cold water whirh removed approximnt~r~ly 10% of activity together with water-soluble inactive material. Subsrquentl,v the organic ph:we (170 ml.) was diluted with 1 vol. ?a-heptanc and extracted three times with 200-ml. portions of cold 0.1 S acetic wid. This served to bring lllc*iferin into the aqueous phase leaving behind waterinsoluble impurities in the organic phase. The romhined aqueous est.r:tct,s (808 1.11.) were extracted with 100 ml. wl>utanol ,vielding a residue with 750 l.u. (15C’, 20 l.u./mg.). Large losses were due to the oxidation of luciferin, resulting in part to the formation of stable emulsions which prw longed the exposure to air. Ncvert helcss, this procedure yielded, without the use of a mineral acid, the best preparat,ion of crude luciferin rcl:itively free from large quant it,ies of polar and now polar impurities.
Early dialysis experiments with luciferin preparation ohtuined by solvent fractionation, Method I, in water ml&r nitrogen nt’rnosphere with two types of acllophalle nmnhranes (Visking cellophane dialysis tubing 4-f:s2 and 23 / 32) established the escape rat,es with half-times (t/z, time required for the escape of 50 5; material) l,et~v-rcn 3:3 and 36 min. The esperiments were patterlied aft’cr the work of Craig and Icing (lo). Dialysis was followed hp weight, optical
density, and luminescence mcasurement,s. On the basis of t,he comparison of the rat,c curves obtained wit’h t’hose of other known compounds obtained under similar conditions, it’ was inferred that the molcculal weight) of luciferin is Mweeu 300 and 500. In a later experiment, a sample (70 1.u.) of the luciferin prcparat.ion obt,ained by solvent fractionation, Method II, was dissolved in 0.5 ml. of 0.1 %’ ;“\;a&Q solution in water (pH 4.5), centrifuged, and the supernatant was dialyzed at 20°C:. in an analytical cell wit,h acetylatcd 1852 Visking cellophane tubing against, 10 ml. of the same solvent. The dialysis was performed according to the met,hod of Craig, King, and St,racher (I). The half-t,imc of escape (t/2) t,hrough the membrane was 230 min. w&h a total rcco\:ery of 10.73 1.11.(14.8 ‘I;). With an unmodified I$/32 membrane a similar sample (70 1.11.) in 0.1 ‘i; XaBz04 under the same conditions gave a t;‘2 of 125 min. with a recovery of 66.46 1.11.(95 %). Subt’ilin (4.5 mg., molecular weight, 3200, 0.8 absorbnncy units,‘mg.,’ ml.) was taken as a reference compound giving in the same solvent with 1552 membrane t/2 366 min. with a recovery of 0.34 absorbance units at 280 rnp. In tbe second series of experiments, 0.01 N acetic acid was used as a dialyzing medium. With a sample (38 1.1~) dialyzed through 1932 membrane at, 23”C., t,/2 was 103 min. and t,he recovery 37.7 1.1~ ((3070. Under similar conditions subtilin (5.8 mg.) TABLE RATES (tlS DIFFERENT
Ihr,us~s
min.)
‘9*,2
membrane Substance 0.01 s acetic acid
Luciferin Subtilin Luciferin
+
pa
p:tin HCl-treated ferin Itedialyzed ferin
luciluci-
I
OF LUCIFERIN COXDITIONS
UXDER
acetylated ‘562 membrane
had a t,!2 of 80 min. with the recovery of 0.834 absorbance units at, 280 mp. Wit,h an acc%ylated 1q2 membrane and 0.01 N acetic acid, a lucifcrin sample (60 1.11.)gave t ‘2 400 min. at 22.5”c1. with t,he recovery of 50 1.11.(83 ‘X). It, is evident from Table I t,hat for the same preparat’ion of luciferin t,he nat’ure of the membrane and the dialyzing medium have a pronounced effect on the magnit,ude of t/ 2. httfmpts were made to hydrolyze this luciferin preparation with papain and t,o dialyze the resulting reaction mixture to see whether t)herc was a change in the escape rate due to the treatment’. A sample (62.4 1.11.)was dissolved in 1 ml. of 0.01 ‘;. cystcine solution in 0.067 A1 phosphat,e buffer (pH 6.8), reacted overnight’ at room temperat,ure with 4.2 mg. of two-t,imes rrystallizcd Worthington papain prcparat)ion, and dialyzed in 0.01 N acetic acid with 18,iZ membrane giving t/2 160 min. with t)he recovery of 21 1.~. (34 ?i ) (Table I). The significant retardation of diffusion rat,e could he due to the association of luciferin complex wit)h the rnzymc.
It was of int,erest to compare the diffusion rate of luriferin treated with 0.5 Ar oxygenfree hydrochloric* arid at) 100°C. for 1 hr. with that of untreated mat,crial. This is the hydrolysis method used by Anderson (6) for t,he drbenzoylation of benzoylluciferin and by Hirata d al. (7) for the preparat,ion of the crystalline lucifrrin. X sample (84 1.u.) subjected to such a hydrolysis with 3.5 ml. of 0.5 S HCl and 0.5 ml. n-but)anol in an evacuated tube and recovered in the form of a residue was dialyzed at, 24°C:. with an l:$z membrane in 0.01 N acetic acid. This gave a t/2 of 25 min. and with t)hc recovery of 71 1.11.(85 96). ITrider t,hcse conditions of acid treatment,, peptide bonds rnight be hydrolyxcd and give rise to smaller, but, still active, fragments of luciferin. To avoid t,his possihilit’y, :I fresh sample (2,500 1.11.)of luciferin cxt~rnct was dissolved in 10 ml. of 10 R) methanol in 0.1 N arctic acid, placed in ly$z tubing and dialyzed at. 5°C. mldrr pure nit,rogen against, five loo-ml. volumes of 0.1 A’ acetic acid. After this no activity was found in the dialyxatc. I’ndrr t hew conditions the t/2
was :jOOmin. with t,he cluantitativc re~o~ry of activit,y. The dialyzntes were combined, nlld usrd for corltitc,r.currcilt w:tporatrd, distribution as dcscrihcd below. A sample (20 1.11.)of this material was dinlyzcld in an analyt~ical cell (4) with an l,@$z membrane in 0.01 S acctia acid giving a tL? of 10 min. with the rcco\-cry of 17 1.11.(85 ‘Y). The first, dialyxatc (10 ml., G.8 1.u.) was used t,o study thr spontanrorw loss of activity during the timcl of dialysis (64 min.). It was rcdaccd to 6.2 1.~. (!)I %). The result of this cxpcrimcnt indicates that the first dialysis NYLSsufficient to rcmovc a cwnplcxing irnpurity rcsponsiblc for the initial slow diffusion rate of lrwift~rin.
activity of ~65 l.u.,/mg. indicatjing a purification of 6000-fold over the dried organisms. Thr cut shown (tulws 8-23) \vas takrn fol rccowry. The absorptiou spectrum of this mntcXrial ill mct~hanol shown in I;ig. 1-,was similar to that rrportcd in thr litwaturc (3, 5), hut’ obviously partial oxidation had alwad> taken pl:wc. Amino acid analysis by t hc m&hod of 1Ioore, Sparkman, and Skin (I I ) showed insignificant, amounts of arnillo :wid twcpt lysine, wpartic ac’id, glutamir wid. and isolrucine. (‘.CJj.
with
Ed-act
from
diethod
/I1
=\fter investigating sevcwl systems, tht> most promising one wit,h thcl rcsidur from .1fter preliminary trials the extract residue this txtract swmad to he WC made from :I solution of 0.1 ‘:I sodium hydrosulfitc~ (‘onfrom 200 g. of the dried organisms which acitl~n-hlttarlol--ngave 12.000 l.u. was distrihutcd to 100 taining 0.1 4 astir trausfcrs’ at 4” under purr nitrogen in the h~ptanc-mc~thallol (1.8:0.75: 1.%5:0.2 v v). system 0.01 S HCll (containing 0.01 5% Hccauw of tht> knowrl stabilizing cffcc*t of ascorbic a~id)-~y~lohexarl~~?~-l~~lt:li~[)l (2 : YazS.J4, an atkmpt was made to carry out I : I v/v). The phase \-olumrs wcrc :L$ ml. the distribution at 25” in a C.C.D. apparut.us with 1000 tubes (a,?? ml. phase volrmws;). .I Thr~ weight (nftcr correcting for aworbic acid) and activity patterns (in terms of run of 1000 transfers rcquircd se\-cral (1:~~s volts; ml.) :MY shown in Fig. la. The most, of cwntinuous operation. The rcsidlw sample wtivc material was that from tubs SO-70 containrd 2800 1.~. alld weighed ahout 1-M which npprowhcd 59 l.u.,/mg. or about w. 9X-fold purifkat ion from the dried organAnalysis was made of both rlppw and lower phases try luminescent, activity and isms. ,&Jut half the total light units origillall>’ talwrr could h awountcd for in the hy optical density at 4% rnl.c. Only two pat tern. regions of activity n-we found lvhicah COYTn~o cuts were takrn as shown in l’ig. la rrspondrd to thr opt,ical density hands for rwovwy. The rrsiduc from cut, 2 (15.8 shown in F‘ig. 3. However, t.he hands at 1.11.‘mg.) was redistributed under nitrogrn t,ubw 325-400 had much higher acativit y at 1” to 100 transfers in the system 0.01 N than the hand at, 50-I 00. The total rrcwvwy HC’l-ethyl :Icrt:ttc-n-~)utanoI-mcthallol (3: of light was poor, ahout 563 l.u. or 20 “; of :I:O.25:0.5). This gave the pattern shown in thr starting sample l+?g. lb. The first band had less specific The material in the most, active t)and, acti\-ity (20 1.11.‘mg.) than the broader t nhes 535420, was rcwwwd by rl-aporation scc~~ntl hand (!I I l.u.,t mg.) which uworurtjcd in a rotatory rvaporator, taken up in 50 nil. for 75 “; of thr activity. Howwr, in all only n-butanol and rxtracted t)hrec timrs wit,h 30 ahout one third of thr total activity was ro- ml. watrr t,o rcmow inorganic: salts. The co\-cwd. organicfi layrr gave i.2 mg. rrsidw which .I ccwtclr cut (tubes 30-50) from the I\-idcr still had :L sprcific activity approximating halltl VW rwo\wcd and rcdistributcld ill the Cl 1.u.’ rng. and showrd thr typical ahsorpSNllf’ s.vstcm rmdcr the snmc conditions. t ion specl ruin of partly oxidizrd IliciferiIi This gnvc pattwll lc. Only one active lland (3). was now olwrwd with a K approximatirlg Part of this material was hydrolyzed in (j that of t ht> first hand in the pwwding pat2V IICl and analyzed for nrnillo acids by the tcrll. ‘I’hc> most wtivc frac+on had a spwific nwthotl of Aloow, Spwkman, and Stciin (I I.). (‘.(‘./I.
with. E&act
from
AI&hod
II
12 2 10
9 P ” 0.0
FJ$ .c x 6 .‘j: ; 2 4r 5 : 2 .-2 E
0.6
i
2 0
20
40
60 Tube
80
20
100
40
60
Tube
No.
60
No 6)
(a)
365l.u./mq. I
HI
I
Iy,LvL^hl
;
100 40 60 80 Tube No. Cc) FIG. 1. (a) C.C.D. patterns with an extract from Method II; (h) pat,terns from redistribution l.u./mg. matjerk from (a); (c) patterns from redistribution of !)1.2 l.u./mg. material from (h). 20
with Extract from Method Membrane Di$usion
of 58
The t#ot,alrecovery of amino acids accounted for only about, 23.57~ of t)he sample. All the
C.C.D.
common
Since the material active with luciferast after passing t,hrough t,he membrane had different properties, a distrihlrt8ion was at-
amino
acids
except
were present in ratios covering not more t,han 1 to 5.
tryptophan 51 spread of
iII
after
CTPRIDIKA
Wave length FIG. 2. Absorption
spectrum
curve
307
LUCIFERIS
of material
- rnp recovered
from
distril-xitil
r
n shown
in lc
I
-1
- 2.4
0.6 I 0.5 $
0.4
3 2 Fi d
0.3 0.2 0.1
100
200
Fra. :S. C.C.11. pattern
300
400
500 600 700 800 Tube No. from 1000-transfer distribution of extract
900
1000
from i\Iethod
TIT.
308 z
0.40
T 2 + 0.30 ii Ek 2 0.20 $ 3 0.10 t’ a Fi d 40 FIG. 4. C.C.1).
80
120
200 160 Tube No.
pstt2ern of material
alt,hough it was considerably more narrow t,han a calculated curve. Recovery of this band gave a few milligrams of residue with t,he typical absorpt,ion spect,rum of partly oxidized luciferin. After hydrolysis, amino acid analysis by the ionexchange met,hod of Moore, Spa&man, and St’ein (11) show-cd only traces of serine, isoleucine, and lysinr: tot,aling not more than 2 7%of t.he residue hydrolyzed. EXPEHIMEXTS
WITH BETYZOYL~TEI) I,r-ClFERIiY
A mcthanolic extract, of defatted (“yppowder was evaporated to a residue and the residue suspended in n-hutanol. After centrifuging off t(he insolttblc nMeria1, the solutjion was used dircct,ly for henzoylnt,inn hy the method of iZnderson (6). For example, a Xl-nil. solution containing approximately 6000 1.11. in n-but,anol was renct.ed at 0” tmdcr vigorous shaking with t.hree successive 1-ml. port.ious of benzoyl chloride added dnriug 1 hr. The solute was now found not to he able to luminesce in the st,andard test with Iucifcrasr. After standing an additional hour, the solution was treated tvith 50 ml. water and ext,ract,cd with rt.her. The ether cxt.ruet, was concent’rated to aboutj %5 ml. and diluted w%h prtroleum ether until no activity (following hydrolysis as given below) remained itI the solution. The active precipitate was then taken up in ethyl ether and ext’ract’ed \vit,h 5 ‘/ XaHC’Oa followed by 10 % acet,ic
ridiaa
from hlet.hod
240 III
after
280
320
dial,wis.
acid. The henzoylatrd material was ucutral and remained in the ether. The oily residue obtained on evaporation of t,he ether could be reconverted t.o a sttbstance which gave light’ with luciferase hy treating it with 0.5 :V HCl and n-hutanol in a sealed evacuated t,uhe for I hr. at 95100”. Before evacuation the oxygen in the tutu was replaced by pure nit,rogen. The rwovcry of act,ivity seemed to range from 25 to 50 ‘T; of t,he original. ;\lemhrane diffusion (-2) experiments were made on t,hc hrnxoylnt~ed residue. In contrast to the active unhrnzoylated extract, tho fwnxoylatcd material was not soluble in water. The diffusion cxpcrinw1t.s were, therefore, made irr 7.5 5: aqueous methanol at room temperature. With 19;;~ Visking most of the activity (measured after hydrolysis) escaped with a t/2 of -20-55 min. Even after 18 hr. ahout 10% of the herlzoylated activity did not diffuse through the membrane. 1Vit.h the more porous 2352 Visking, however, all the hcnzoylated activity readily diffused out. The ryi2 membrane, therefort, gave definite fractionation. A large sample of the benzoylatcd prcparat,ion was frwtionated by dialysis through lY&( Visking and subjected t,ct C.C.D. in a system made from nlethallol~c’y”lohexane-ethyl accbtc-water (2.2 : 2 : A : 1 1,/v). In a 1000 tube 2,‘2 ml. machine, l13A transfers were applied. The pattern oht,ained is shown in Fig. 5. At intervals across the pattern material was recovered, hydrolyzed a,nd
2.0
1134 T
0.29 (
-I
0.40
1.6 v--4 F 1.2 N 2 R 0.8
0.12
0.06 t
0
0.4
0
100
zoo
300
400 500 600 Tube No.
‘700
800
900
ii8 0.20 gCL 0.10 11000 0.30
310
MARFEP,
CRAIG
Indeed, hydrolysis may have occurred hut the smaller molecular size of the acti\-it,> was clearly indicated by recovery of act,ive diffusate through I,8 iy Visking cellophane in 0.01 :\: acetic acid mit’hout. prior hydrolysis and thcll rcillvest,igat,ion of it,s diffusion rat,c. As can be seen from Table I, diffusion now was even mow rapid t,han with the HCltreated preparation. The effect. of the membrane diffusion in removing luciferin from the material to which it was hound was further shown by the difference of the original cxt,ract and the diffusatc in countercurrent distribution studies. Moreover, the smaller size of t!he luciferin was also shown by mctnhranc diffusion studies wi-ith the twnzoylat,ed lucifcrin. Here, hecause the benzoylat,cd derivative was not soluble ill wat,er, i5%, methanol was used. This is known (10) to ret,ard greatly the dialysis rat,c of various solut#es. &uit)e apart from the membrane diffusion studies the c~ounttrcurrcnt distribution studies indicated a strong associat,ion of the activr principle t’o inactive material. This was shown by the fact that, a K determination in a suitable system on the basis of luminescent activity with a crude extract, would show a considerable shift in K with hut’ a few transfers. The succrssivc distributions shown in Fig. 1 with different systems indicated a major problem to he that of disengaging thr active principle from the peptide material to which it was bound. Figure 3 is equally ii&resting in this connection. Herr an attempt was made to make a prolonged distribution at room temperature in the prcscnce of t,he known stabilizer (9), sodium hydrosulfite. After 1000 transfers the majority of t,he remaining activity was in a single band which seemed to behave almost ideally frotn t,hr standpoint of countercurrent distribution. However, the material in t,his band did not have the specific activity expected, and amino acid analysis after hydrolysis indicated nearly at1 the known amino acids to tw present. Evtn to distribute in this way in such a nonpolar system would rccglirc that t,hese amino acids arc combined to form a molecule wit’h no effective polar groups. Evtn t,hc cyclic an& biotic polypcptidcs devoid of basic or acidic
AND
HARVEY
groups have shown more polarity than these substances. The experience here is nnlch like the chromatographic expericncr of Tsuji ct al. in attempting to isolate C’ypridina luciferin in pure form. Chromatographic: experiments in this laborat,ory also gave rcsult,s sitnilat to theirs. Apparently the conditions used did not have suflicient dissociating st,rengt,h t,o permit separation of the active principle from the accompanying peptide material. When similar tnatcrial was studied except that it, had dialyzed t’hrough 1yi2 Visking cellophane, the system used in l:ig. 3 was no longer suit~ablc. A more polar system was required, and the result shown in E’ig. 4 was obtained after ?,-I4 t,ransfcrs. The single act,ive band was not ninhydrin positive, and after hydrolysis amino acid analysis showed them to be almost cntirrly absent. This experiment, in spite of ot,her experiments to the cont,rary suggests that the active principle in the gland is a relatively small solute which can tw dissociated and separated from pept’idc mnt,crial of very unusual character. The active principle still can tw of cyclic nature derived from amino acids. While all our cxperitncnts with free Cypridina luciferin were ronsist,ent with this view, those wit’h t,he henzoylat)ed mat’crial were not. The t)enxoylat,ed material distributed as a very c*omplcx mixture as pattern in Icig. 5 shows. The partition ratios across the chart, (comparison of upper and lower optical drnsitics) arc consistentJ with t,he tube numtwr in which the solute occurs. Hydrolysis and luminescent asslty indicated activity scattcrcd at1 across the chart,. The numbers ahovc t,he curves give the l.u./‘mg. (lomplctc hydrolysis of the material in sevcral rrgions and paper chromatography indicatcd numerous ninhydrin-positive spots corrwponding to amino acids. The cut of tubes 350-425 was recovered and rcdist,ributcd tmiw to 120 transfers in the system glacial acetic acidPwatcr-cyclehcxanf-met hanol-chloroform (2 : 1 : 1: 1 : 2). This gave a single baud of about theoretical Ividth, but on hydrolysis and lumincsccnt. assay the activity was found to be increased only to 0.82 l.tl./mg. The hcnzoylation experiments taken alone
C!TPl~ll)IS.1
wem to ildi(xt(~ that (‘~ypn’dina lucifcrin is an activr c~hromophorc c~onjugatcd to LI hetcrogcncow spwtnun of pcptide materisl. This seems to Ix supportctl by other fmct,iolintion rxperimcwt,s inchidilig various forms of chromatography. While the henzoylatcd matrrid ww mwh more stahk than the frrc lwifcrin, it also W:LSfound to low slowlp the ability to lumincsw with lucifcri~l following hydrolysis.
LTTCIFEIt
IN
::I 1