Isolation and characterization of phospholipase a from sea snake, Laticauda semifasciata venom

AH(‘HIVb:S

OF

BIOCHIZMIS’I’HY

Isolation

and

AND

HIOPHYSI(‘S

140, 96-106 (1970)

Characterization

Sea Snake,

of Phospholipase

Laticauda

Semifasciata

A. T. TU, R. R. PASSEY,* Department

of Biochemistry, Heceived

Colorado April

AND

I’.

State University,

29, 1970; accepted

A from

Venom’

1%.

TOOM

Fort Collins,

Colorado

80521

June 1, 1970

Phospholipase A has been purified from venom of the sea snake I,alicauda sewuz’jasciata by chromatography on CM-cellulose followed by chromatography on DEAFcellulose. The preparation, which was shown to be homogeneous by tonal electrophoresis, ultracentrifugation, isoelectric focusing, and gel filtration, had a specific activity seven times that of crude venom. By using ovolecithin of known composition in the 1 and 2 positions, the enzyme was shown to be specific for the 2 position, liberating mainly unsaturated fatty acids. The molecular weight of the enzyme, determined by bot.h Sephadex gel filtration and by a combination of s and 1) from ultracentrifugation is 11,000. Of several phospholipids tested, only phosphatidyl choline was hydrolyzed. The enzyme was activated by Ca2+ and to a lesser degree by Mg2+. The cat,ions Zn*+ and Cd2+ both complet.ely inhibited the enzyme, even in the presence of Ca2+. The prcp;trat,ion was most active at pH 8.0, and at temperatures between 35 and 10”. The enzyme exhibited hemolytic act,ivit,y which was greatly intensified by the addition of lecithin. The purified phospholipase A was nontoxic, nonhemorrhagic, and exhibited only slight myolytic activity.

The ability of snake venoms t,o cause hemolysis has been observed by many invest’igators. Lysolecit#hin, produced by the action phospholipase A (EC 3.1.1.4) on lecithin, has been established as one of the causative agents producing hemolysis (1). Phospholipase A has been found in a variety of mammalian tissues (‘2), as well as in the venoms of scorpions (3), bees (4), Gila monsters (5), and snakes. Although numerous studies have been undertaken t’o study the specificity of snake venom phospholipase A, few have been carried out, with a purified phospholipase A preparat,ion. Wu and Tinker (6) recently isolated a phospholipnse A from Cwtalus atros (Western diamondback rattlesnake) venom, while Wells and Hanahan (7) isolated two forms of phospholipase A from 1 This investigation was support,ed in part by USPHS Grant, 2 It01 GM 15591, and NIH Career IIevelopment Award 1 K01 (+M 41768. 2 This paper is based on a dissertation suhmitt,ed by Ii. B. Passey in partial fulfillment for the degree of Ph.]). alt Colorado State IJniversity.

C. adanlunteus (Eastern diamondback rattlesnake) venom. Salach et al. (8) found nine different phospholipase A act.ivities in Nuja nuja (Indian common cobra) venom. As compared to land snake venoms (l”amilies: Crotalidae, Viperidur, and Elapidac), sea snake venoms (Family : Hgdrophiiduc), are much simpler in composition (9). Aloreover, very few chemical investigations have been undert~aken on sea snake vtwoms. The presence of phospholipasr A in sea srdw venoms has been observed by Carey and Wright, (10) and by U\vatoko et al. (11). However, no at,tempt has been made to isolate phospholipase A from thrw venoms. This report will describe a two-step prow duw for the isolation of phospholipaw ~1 from sea snake venom, and repori on sornt’ of its propcrt ies. MATF;II

IALS

ANl)

METITOI)S

Materials. Venom from the sea snake I,a/ic,aurla semijasciata (Reillhardt ) from Amami Island in thp ICast China Spa. wt~s IISP~ RS thr SOIII'W of phos-

PIIOSPIIOT,TPASJ~

.4 FltOhI

pholipase A. The venom was collected during the summer of 1967 by A. T. Tu and I’. RI. Toom. The venom was gently squeezed from ihe glands, lyophilized, and stored at, 4“ nnt il fractionated. Tritou X-100 was purchased from Sigma Chemical Co. Cellex-CM (C&Z-cellulose) and Cellex-1) (1>fS~4T~-ccll~1lose) wcrc p11rrhasctl from Rio-Ilad Lal1orat,ctrics and prepared ac~rding t.0 lhr m:\n11fwctuers suggestions. Sephadex G-75 and (i-10 were obtained from Pharmacia Fiue Chemicals Inc. Standard proteins for t,he molec1dar weight. det,crmination utilizing Sephadex were obt,ained from hIann Itesenrch Laboratories. The Ampholine carrier ampholytes and elect,rofo~~1sing col~mut nsed in the electrofocusing proc’ednre were purchased from LKTS T11strumcnts. Lysolecit.hin was obtained from Pierpe Chrmical Co. I,-cu-Lecithin (synthetic was pm&ased from iX11tritionnl Biochemical Corp. The fnllowing chromat.ographically pure substances were obi,ained from General Biorhemicals: eerebroside (beef brain), sphingomyelin, phosphatidyl et,hanolamine, phosphstidyl I>-serine, phosphat,idyl iuttsitide (fraction I from beef brain). e1lr~lioti~~i11, phosphat,idic acid, I,-cu-lecit,hin (beef brzlin), and lysolecithin. Lecithin (pt1osplioi.idylcl~oiine) was lmrchaseti from Mann ltesearch Laboratories. The preparation was p11rifieI.l by passage through a column of neut,ral alnmirra arcording to the method of Wills and lra11at1a11 (7). The p~1ri~e~i le~,it.hi11 was then precipitated twire from a mist ~IPP of warm arcmic-methyl et,hyl kei one (1: 1 v,.v) (12). The white powder thrls obt,aitted appeared homogene011s after thin-layer ~hron1atogr:q~h)on silica gel using cl1lorof~~rrr~-mctl~1~t1ol-waler (95:35:-1-). The fatty acid compositiort of the l&thin was determined by 1~le~~t1a~~ol~sisand gas-liquid chromalograph.y (13) The fatt.y acid compositio11 of the lecithin, as shown in Table III, WRS no1 signifirantly 1lifferet1~, from that reported t1,v Mrnxcl and olcott~ (14). TsoEal,ior~ procetlure. Previously washed imd qhargcd C?tGcellnlose was eqnilibrat.ed with the init,irtl buffer, 0.01 M phosphate conteini11g O.OOl M 11:1IT.4 at pH 0.02, for 21 hr. The final colrmu1 bed size was 2.8 X 39 cm. The cc1lnm11 was loaded with 0.500 g of the crude venom which had been dissolved in 5 ml of init,ial buffer and centrifllged. Tt1c flow rate was adjr1stetl to 15 ml/hr and 3-ml fraci ions were collected rising an ISCO model U-A2 rtlt raviolet. analyzer. The fracf io11 which was positive for phospholipase A was desalted with Sephndex G-10 lgophiliaetl, and dialyzed against 0.001 M Tris b11ffer, pH 7.70, anti then chromntographed 011 a DEAN-cellulose colrmm previonsl) eql1ilihrated wit.11 0.001 M Tris bl18er, pII 7.70. The ptrifkl I~l;~)spholi~~as~ A from the ~~,4~-rell11tose

SEA SNARI:

VKNOhl

col~rrnrt was lyophilixed, desalted. n11d si.ore+l at, 1’ in :I lyophilizcd st.ate. ctllracentrifugation. The spi IlCO Anulyticaz model E ult,r:leentrifugeI with an AN-I) rotor, au Kpon double secttir, and an Epon d(~l~t)le-se(~~or capillary syrrthet ic bou11dary cell were used tn meas11re the nerlim~fit ation a11d difY11sion roefGtG.f~ts. The [ti~~llsio11 111e1~s~lrcn~~il~swere made al. 9915 rpm with R phospholipase A solutiou of 10.0 mg in 1.0 ml of 0.100 11 Tris, pII 7.00, dialyzed against> t.he same bufl‘rr for 1F hr at 4’. Phr1tographs were t t&ken of the Schlieren pat,lerrts at. +min intervals for a tnt.al of 40 min. The physical measurements were made with :t Kiko11 profile projector, model tic microror11~1xrator. The sedimentation coefGieiit, m~as11rcments were made at 59,780 rpm on ttie same solut,ions as used for iht’ diffusion 11~e:~s~1reni~?11~s. Phnt ographs were taken at, X-min intervals for a tot,al of 2 hr. PllI cxpprimerits were carried out at 20”. lsoelec~tric fomsing arktl pol,yncetnle elwtrophrwesis. Isoelectric foeusing was performed in a 1%ml col~mu as desc*ribed previously (15). Zone elrctrophoresis c~sp~ritneirts were carried 01it, on Gelman ~)oly:~~et~~~,e strips. The buriers used were: 0.028 y barbital, pII 9.1; 0.01 M acetate bluffer, pll 5.0; aud 0.01 M phosphate cSolltainillg 0.001 M ISDTA, pII S&G. The strips were srlbjectcd IO elect rophoresis for 2 hr ai, 200 V (1.5 mA per st,ri@). At the c11d of 2 hr. t,he strips were stained in 0.25’; lS11fi:tlo black dissolved in mrtha11ol-acet in acid.walrr’ (4:4: 1 v/v.‘v). The strips were then washed clear of the St ai11 in aohyctro~1s methanol, at1tl cleared in 10:; acetic acid. :l~tiino acid (~~~~l~?f~~~~. Amino acid analysis ~-1~s performrd acc~ording t 0 the procedlire of ~Ioorc and Sit611 (l(i). Hydrolysis w:ts performed at 110” jl 1” for 22 and 70 hr. The analysts were carried ant on a Beckman amino acid analyzt~r model 120 B. For the a11alysis of cyst,rine a sample of 1.73 mg of phospholipnse A was lrrnt et1 with performic arid :~~.(,(~r(li11g to the m(ht hod of S&am e( (if. (IT) prior 10 hydrolysis for 2O trr as drscribrd ahovr. Bnzyrrie acticity. Pt1ospl1~~li~~:1se A :tc*tivit y was rncassi1rcd bq following the rate of delivery of 0.0018 N NaOTI necessary to keep thr pEI of thca reartioit mixture constant, 11s11ally at plf 8.00. This was a(,t~ot~~plist~e(I by t1sing a l
TU, I’ARSEY,

9s

ANI)

TOOhI

IO NaCl, 0.22 g/100 ml C&Cl?, and 0.037 g/l00 ml BUFFER IF BUFFER 2 +---I BUFFER 3 --y EDTA. When no Ca2+ was used in the reaction mixtures water was used in place of this saline solution. The reaction mixture was stirred vigorously with a magnetic sl irrer and maintained in a Y nitrogen atmosphere at 23”. Reaction products a E5 were separated on silica gel thin-layer plates using t2 t.he solvent system pet,rolenm &her (~~-~9 bp), 4” diet.hyl ether, glacial acetic acid (85: 15:l). Quantitation aft,er esteritication of the fat,ty acids with (lin~eth~~ls~llfate was carried out by means of gasliquid c~~rornat,o~rap~ly (13). I)uring the pttrifica100 200 300 400 500 lion process a slight modification of the egg-yolk TUBE NUMBER clearing method of Marinetti (18) was followed. Egg yolk w&assuspended in 0.05 M 2,~,~j-t,rir~~eth~l FIG. 1. Shown is a, typical CiV-cellulose chropyridine buft’er containing 0.005 M CxCi~ and 0.012 mat,ogram llsirig 500 mg of I,aiii:aztflala semiM N&l at pH 6.5. The suspension w-as centrifuged fasciala vRnom, c,olumn size 2.X X 39 cm wit.h a to remove large particles of egg yolk and clearing flow rate of 15 mlihr. The column was e~~~ili~~r~~te(l of the suspension was followed at, 700 nm. with 0.01 M phosphate buffer with 0.001 M IOTA :tt Biological uctivily. Toxicity tests were con- pH 6.02 which was also used as the first buffer. ducted by injecting 0.20 ml of verwm fractions into Buffer number 2 was 0.03 M phosphate wit,h 0.001 t,he tait vein of 20-g Swiss White mire. Five mice M EDTA, plI 7.72. A gradient was established by were used at, each dosage level and tht: number of adding buffer 2 to 200 ml of buffer 1. The dot ted dead mice were observed after 24 hr. line represents the gradient. nuffer 3 is 0.03 M I~eInorrhage was de~ernlined by s~~h(~~~tat~eo~~sly~)hosph~~t,e,0.001 M EDTA, anti 1.0 M N&I, pTT injecting 0.050 mg of phospholipase A dissolved in 7.72. The gradient was established by adding 0.20 ml isoi.onic saline into Swiss White mice (19). buffer 3 to 200 ml of buffer 2. Absorbancy at 280 After 21 hr the mice were sacrificed, the skins re- mm is plotted against. tube number. Three-millimoved and examined for signs of hemorrhage. liter fractions were collected, The hatched arra Myolysis was determined by injecting 100 pg represents phospholipase A activity. of phospholipasc A dissolved in 0.10 ml isotonic saline into the hind leg thigh muscle of Swiss sernifasciuta venom. After fraetionatJian on a White mice. After 24 hr t,he anirnala were sacrificed Cl&cellulose column, the active fratct,ion and t,he muscle fixed in lOc;bformalin. Histological was further purified on a column of DEAEslides were prepared and stained in helnatoxy~in is ilfusand eosin. Photomicrogrephs were taken of en- cellulose. A typical fractionation t,rat,ed in I>igs. 1 and 2. The various active zyme-treated and control tissues, The amount of phospholipase A and crude fractions were desaked using a Sephadex Gvenom needed to cause hemolysis of 507; of an 10 column and deionized wat,er as the eIut,ing erythracyte suspension was determined using solvent, lgrophilized, and stored at, 4’ until Swiss White mouse erythrocytes washed t,hree used. The fractionation was independently times in the tSrimethyl pyridine buffer used in t,he repeated four times with the same patterns egg-yolk clearing test. The final concentration of and yields. ,4 summary of the purificat’ion erythrocytes was lo7 cells/ml. The test was consteps is preaent,ed in Table 1, where it can IX: ducted hty incubat,ing 1.0 ml of a solution of leciseen that the specific ~~~os~~~ol~p~~se A Xt bin (0.1 mg,‘ml) with 0.05 ml of the enzyme prepativity increased about ‘i-fold. The recovrry ration for 10 min at 37’. One milliliter of the of protein (30 mg) represents about (i % of erythrocyte suspension was t,hen added, and the mixture was incubated at 37” for 30 min more. The t.he st,arting material. reaction mixt,ure was then centrifuge 1 at 800 g and Criteria of l)wity. Aft,er polgacet,ate ekethe supernatant, fluid decanted. The absorhancy t,rophore& at pN 9.10, 12 component,s could at 410 nm w:ts measured, and t,he 509; hemolysis be det,ected in ur~fra~tio~lated venom, three point (‘f1Um) was determined by f he met hod of components in fract,ion C.\I-V, and only :t I,it,chfield and Wilcoxon (‘LO). single component after t,he enzyme had been

RESISLTH

purSied on both C~\[-~~~llulos~~and DEAEcellulose. To further rnlablish t’he purity of phospholipasc A, buffer systems of pF1 7.(iO, 6.04? and 5.00 ww :~lsc)us;r~tl. Tit ~a& irl-

JFFER IbiWFFER 2-A BUFFER

3 ------.I

BUFFER

stance, a single band migrated slowly t ownrtl the cutllodt:. Isoelectric focusing (pH S--10) of unfractionated venom revealed 14 compownts; only one of which contained phospholipaw A in Fig. 8, \vhtw xtivit~y. As 1~x1 br Swn purified phospholi~):~se A xw subject t~l to isoelectric focusing undw the same contlitions, only 0Iw ptxlr was rvidcnt, :IrItl this peak contained the phospholip~st~ A activity. Gel filtration of the purified l~l~osplloli~~:lst~

4-

m

A TUBE

NUMBER

FIG. 2. Shown is a typical l)l’:All;-cellr~losc rhromatogram losing peak V of the CiScellulose colrlmn. The co1unu~ was 1.0 X 17 cm and t,he flow rate was 12 ml/hr. The rollunn was equilibrat.ed with buffer number 1 which was 0.001 of Tris at pII 7.70. Peak V from the CM-rel111losc rollunn was dialyzed against buffer 1 before it was applied to the colllmn. Buffer 2 is 0.03 M Tris, pH 7.00. Buffer 3 is 0.05 M Tris with 0.001 M I<:l)TA at pH 7.70. Buffer -1 was the same as brlffer 3 but with 1.0 ix NaCl added. The shaded area represents phospholipase A activity. Absorbancy is plot,ted against, the tllbr nrlmber. Three-milliliter fract ions were collected.

Fraction

Venom First fractionation, Cbf-I CM-II CnI-III CM-IV CM-V CM-VI CM-VII Recovery Second fractionat,ion” DEAE-I l)EAKII l)l5.415-III llecovery

Total protein (ma)

Specific titration methd6

“I .7 20.0 .i .!)

115.n 13 .7 176.3 x2.0 3X..i

0 0

0 0 .5X. 1 0 0

Activities 9

g$

011

Sepl1:drs

c:-7r,

p’od’“ctYl

only

oIl(:

symmetrical peak in rt~spect~to both 2SO-I1111 :~bsorbancr :uId phosl~holipse .Z activity. Ult r:umIt rifugation of a 10 mg/ml solut ion of puritit~d I)hospliolip;tst, A a1 59,780 rpm was prrformrd three intlrpendrni timw, and in eaclr ciwc, only one pr:ik \v:ts widcnl . I)utx I o the fact thnt boundary format ion MXSnot, optimal (column 1, E’ig. 4) and dw to t ht: high diffusion coeticicnt of this 101~molccw lx wGght, p-01 t+i, an unnmbiguous :wst~ssmcnt, of the homogeneity was dificult. I’/~!ysica/ pa~a~~~eters. l’urifitd phospholipw A from pwparations 1 and 2 wtrt’ used in t1t.tcrmining both the wdimentation cot#icitwt, and the diffusion constant. l’rom corfficitwt~ prtyxmlt ion 1( :t wdimrnt:ttion (s?~,,,.) of 1.92 X 10FK st’c VW obt:Gled, while prtqxarat~ion 2 yielded :1 sediment at ion coefficient of 1.!13 X 10plK. IMfusion corfiGents (&,,,), d et)t~rminctl from :L solution of 10 ma/ml of pl-wified phosphilipzwc A dis-

.ioo

1

0 0

0 0 1360 0 0

402.1 40.7 6.0 :to 0

0 0 1.X 9

0 0 3700

‘1 Micromoles N&II added/mg protein/mill. b .4bsorbance change at 700 nm/min/mg X 1000. r The CMC column was loaded with the soluble protein after cent,rifgation of 500 mg venom. rl The T>EAE column was loaded with 79.1 mg of CiWV fractiolt from t.he first fractiollntioll.

PH

FIG. 3. Isoelectric forusing pattern of 10 mg of pllre phospholipase A using an ampholyte bldYe1 wit,h a pH range of 3-10. The phospholipase A had been allowed to focxls at equilibration conditions for 48 hr. The peak was active elsewhere in the elut.ion pat,tern. Ahsorbancy 280 nm is plot ted against, the pII of the ellltent from the colllmll.

FIG. 4. Schiliern images of a typical srtlim~~~ta~.ion cocflicient dekr.rnin:ttion are shown. The pic(urcs were taketl, at, a ti0” bar angle, at 8, 32, 50, and 80 mirl after mnximrlm stwetl was reached. The holuldary was l~rcp;td hy me:111sof a synthetic t)ollnd:try tlolthle-secbr cell llsirlg a 10 mg/ml solut.ion of phospholipasc~ A in 0.100 M Tris l)~~fi’c>r, pll 7.00, which hat1 bwn dialyzed

/

:qpiirsl

lhe same hrdfer.

I

3.6

3.8

40

4-2

LOG OF MOLECULAR

4.4

46

4.8

WEIGH7

FIG. 5. Plot of log t,he molecular weight against elution volume for determination of moleclllar weight. Sephades G-75 was previously eyuilibrated with 0.05 M Tris buffer with 0.10 M KC1 at pII 7.50 for GOhr, packed into a 1.5 x 7%cm column, and allowed to eqllilibrate for 36 hr. Void volume (l’o) was determined with each prot,ein measuring the ellltion volume of blue dextran added to the protein solution. The elutions volumes were measrlrrd by a continuous monitoring of t,he 280-nm absorbance of t.he column elilent. The not,ations Itsed in Fig. 5 are given as follows with theil molecular weights in parrnthcscs : 1. ~lrlcayon (3,485); 2. phospholipase A from preparat,iorl 2 (10,400); 3. phospholipase A from preparation 1 (10,200); 4. ribonuclease (13,G80); 5. t,rypsili (23,800); G. chymotrypsirl (25,000); 7. ovalburnill (45,000); 8. albumin (bovine) (67,000); 9. pepsiu (tlimer) (71,000). solved in :md dirtlyzed against 0.100 M Tris, pH 7.00 were, 1.41 X lo-” cm2/scc for both prqmrations. The molecul:lr \\-+$I wns detrrmined by :L

cc~mbination of s :trld U by mc‘ans of tllr Svedberg equation. IJsing v:~lurs of 0.71 for t ha p&al specific volume (ctdculst ed from 111~ amino acid composition by the met hod of Cob :rnd F:ds:rll (Zl)), S.‘,,,,r of 1.93 X 10-l” see, :lrld fI~O,,y of 1.41 X lo-” cm2/sec, :\ moleculilr lveight of 11,000 W:L~ calcul:ltcd. Iising tht mrt hod of Andrews (22) I I)(: molrcul:ir rveight of the purified phospl~olipas? &I w;Ls (1stimatcd from Seph:&;x gL;(‘l filtmtion. Bs c:tn be seen in l:ig. r,, :~n cllut ion volume corresponding to n molecul:tr \vc:ight, of 10,700 WNS obt:lined from prepar&m 1 while preparation 2 gave an c>lution volume: corresponding lo :I molt:cul:U Wigllt of

10,400. The :unino :icid composition of phospholip:ts(s A is pr(hsented in T:Lble II. E&1 figurtb reprt9clnts t,hc :tvrrtLge of six dotcrminat ions. Cvstcint \vas determined :IS cystc,ic :Lcid :&er oxid:ltion \vitll p of substrate concentration OIL veolocit,y of thr enzymatic re:iction is shown in IGg. 0. As cm bc noted from the tigurct, the ratr> of

PHOSPHOLIPASE TABLE AMINO

Amino

acid

Aspartic acid Threonine ,Serine (:lutamic arid Proline Glycine Alanine x Cyst.ine* Valine Methionine Isoleucirre J,cucilke Tyrosine Phenylalanine Lysine II ist,idine rlrginine Tryptophanc Total

103.0 5.5 0 47.8 102.0 33 ,6 56.4 46.6 107 .:3 x.2 x x.7 27.1 4.i.l 134.0 x2.2 X0.7 24.6 46.6 12.3 1000.9

SEA WAKE

101

VEKOSI

II

1

ACID COMPOSITION PHOSPHOLIPME A Amino acid r/l000 !? protein)

A FROM

OF

Red~“S”“/ enzyme

10.6 6.4 6.6 9.:: 4.7 10.0 7.x 12.5 3 .9 0.x 2.X 4.7 9.7 2 .6 7.4 2.1 3 6 0 ,9

...~~~.~ CLBased on a molecular weight of 11,100. h Measured as cysteic acid. r Measured spectrophotometrically.

Nearest whole number

A

11 6 7 !I .i

10 x 12 4 1 3 .7 10 :3 7 a 4 1 108 ..-

substrate hydrolysis was independent of substrate concentration at sub&ate concentrations greater than 12 m&l (19 mg/ml). Under these conditions, hydrolysis was approximately 6.5% complete within 20 min (8 fig phospholipase A). The rate of fatty acid release was linear with enzyme concentration (Fig. 7). As has been observed by ot,her investigators (6,24) a rather significant lag period was observed in the hydrolysis of the above substrates. Thus, an evaluat,ion of K, , based on initial reaction velocities, is most impractical. However, an estimation of specific acCvities after t,he lag period and under optimal substrate concent,rations are in order. These activities were found to be 160 pmoles/min/mg enzyme for ovolecithin and 13.5 ~mole/min/mg enzyme, for syntjhet’ic, dipalmitoyl lecithin. Proof of site of attack. Egg yolk phosphotidylcholine (25 mg) was dissolved in 2 ml of Triton X-100 (5%). Phospholipase A (10 mg in 0.1 ml) was added and the pH mnin-

4

8

SUBSTRATE

12

16

L 24

20

CONCENTRATION

(mM)

6. Effect of substrate concentration on the activity of phospholipase A. The velocity is in micromoles of alkali added (0.0048 s) per min. The pH was kept at 8.00 under a nitrogen atmosphere. The reaction mixture rontained 5 111~ Ca” and 0.01 g Triton X-100 plus t,he substrate in 2.0-ml total volume. Curve A represents ovolecithin with 1 pg phospholipase A, and curve B represents synthetic dipalmitoyllecithin with 25 ~g phospholipase A. The substrate concentration is in moles/ liter. A radiometer ultraburette, type ABU 16, accurate to 0.001 ml, was used in t)he titrations. FIG.

FNZYME

CONCENTRATION

(99)

7. The effect of enzyme concentration on the activity of phospholipase 9 isolated from Laticauda semifasciata venom is shown. The activity is expressed in micromoles of NaOH added per minute. The reaction mixture contained 5 rnM calcium ion, 0.01 g of Triton X-100, and 20 mg ovolecithin dissolved in 2.0 ml. The reaction was carried ollt at pH 8.00. FIG.

tained at pH 8.00. After 20 min (65 4°Chydrolysis), the reaction mixture was lyophilized to dryness. Thin-layer chromatography revealed that lysolecithin and fatt,y acids had been released. The fatty acid composition of the released fatty acids as well as t,he fatty acid composition of the lysolecithin were determined by gas-liquid chromat)ography (13). To shon that, no fatty acids mere being released by nonenzymatic hydrolysis, a blank containing all rract,ants except phos-

102

TU,

PASSEY, TABLE

FaTTY

ACID

COMPOSITION

OF PHOSPHOLIPASE

AND

TOOM

III A HYDROLYSIS

PRODUCTS

OF EGG LECITHIN

Specific fatty acid (mole %)” 14:o

14:1

16:0

Trb

Tr

36.2

0 Tr

0 Tr

Tr Tr

Tr Tr

Tr

Tr

Tr

Tr

16: 1

18:O

18:l

18:2

20:4

1.4

13.8

34.0

12.9

1.6

0 34.2

0 1.4

0 14.6

0 32.8

0 14.1

0 2.7

10.7 52.9 9.1

1.6 2.8 17.6

4.1 21.7 8.2

58.8 18.8 60.0

22.4 3.6 75.0

2.4 0.0 100.0

7.8

1.5 2.8 23.0

2.6 23.5 6.6

60.2 12.6 70.0

25.8 1.6 88.2

2.0 0.0 100.0

-.

Lecithin standard Total fatty acids Lecithin blankc Hydrolyzed fatty acids Nonhydrolyzed fatty acids Lecithin + venom Hydrolyzed fatty acid@ Nonhydrolyzed fatty acids” yC Hydrolysis Lecithin + phospholipase A Hydrolyzed fatty acids Nonhydrolyzed fatty acids y0 Hydrolysis

59.3 6.2

a Notation: number of carbon atoms followed by number of double bonds. b TR, trace. c ,411reactants except phospholipase A, incubated at 23”, pH 8.00, for 30 min. d Fatty acids hydrolyzed from lecithin by action of phospholipase A. Numbers represent the average of three determinations. 8 Fatty acids remaining attached to lecithin and lysolecithin after hydrolysis with phospholipase A; numbers represent the average of three determinations.

pholipase A was incubated at 23”, pH 8.00, for 30 min. As can be noted from Table III, no fatty acids were liberated, and fatty acid analysis of the lecithin was identical to the lecithin standard. The results of analyses in which phospholipase A was incubated with lecithin, shown in Table III, indicate that virtually all of t,he polyenoic fatty acids, but few of the saturated fatty acids were liberated by the purified enzyme. Also, nearly identical results in respect to the fatty acids released, and the fattry acids remaining attached to lecithin and lysolecithin were obtained with purified phospholipase A and with crude snake venom. h’j’ect of pH and temperature. Phospholipase A was found to exhibit a rather narrow pH optimum, with optimum activity being found at pH 8.0. As can be seen in Fig. 8, no enzymatic activity could be detected at pH values below 6.0 and above 10.0. The effect of temperat#ure at pH 8.0 was investigated t,hrough a temperature range of l-75”. ,4s illustrated in Fig. 9A, phospholipase =Z exhibited optimum activity at 3540”. Although the enzyme was inactivated at 7.5”, it was still quite act,ive at 1”. Figure 9B

FIG. 8. The effect of pH on the activity of phospholipase A isolated from Laticauda semifusciata venom. Activity is expressed as micromoles NaOH added per minute. The reaction mixture contained 5 mM calcium ion and 0.01 g of Triton X-100 in 2.0 ml. The reaction was maintained at the indicated pH using a radiometer pHstat titrator TTTlc by addition of NaOH from an automatic burette.

presents an Arrhenius plot for the determination of the activation energy. The activation energy calculated from this plot is 6900 cal/ mole. E$ect of divalent cations. As can be noted from Table IV, M-o types of effects were ob-

PHOSPHOLIPASE

9 FROM

SEA SNAKE

103

VENOM TABLE

A

IV

EFFECT 0~ DIV.ILENT CATIONS PHOSPHOLIPSX A ACTIVITY Metal ion (mu) Cd

Other metal -

5 5 TEMPERATURE

(“‘2

6 -

6 6 6 -

fx lo-5w’, 9. A. The effect of temperature on the activity of phospholipase A isolated from Laticauda semifasciata venom. The activity is expressed as micromoles NaOH added per minute. The reaction mixture contained 5 my calcium ion, 0.01 g Triton X-100, and 20 mg of ovolecithin in 2.0 ml. The reaction was carried out at pH 8.00 using 1 rg of phospholipase A, and the temperature was varied from l-55”. B. An Arrhenius plot for the determination of the activation energy for the hydrolysis of a fatty acid acyl bond catalyzed by phospholipase A acting on L-or-ovolecithin. The log of the velocity as micromoles of alkali added per minute is plotted against the reciprocal of the absolute temperature. The reaction mixture is the same as described in Fig. 9A. FIG.

served when divalent cations were incubated with phospholipase A. Namely, either a partial activation of t’he enzyme or a complet,e inactivation, even in t,he presence of Caz+ ion. Mg2+ exhibited a partial activating effect,, restoring about’ 25% of the activity observed in the presence of Ca2+. On the other hand, Zn”+ and Cd2+ completely inhibited phospholipase A, even in the presence of up to n lkfold excess of Ca2+. Ni2+ had no act#ivating effect of its own, although it did prevent Ca?+ from activating the enzyme completely. Substrate spec$cily. Of the substrates

25 38 80

;Llgz+ Mg*+ Mg2+ h’Inz+ Mn2+ Mn2+ xi2+

EDTA

-

3 .i 5 5 G? 5 j

Ni2+ :; Co2’ 3 co2+ 5 Zn2+ 5 zn2+ r, zn2+ r, Cd2+ 5 Cd2+- .i

20 20

ON

Enzyme activity (%)” 10

100 0 2.i 0 29

-

11

20

0 32 0 58 0 50 0 0 0 0 0

-

a Activity expressed as percentage compared to 5 mu Ca2+.

of activity

tested, only phosphatidylcholine was hydrolyzed. Of t,he two phosphatidylcholines, ovolecithin was hydrolyzed at a much more rapid rate than the synthetic lecithin containing only the saturated fatty acid, palmitic acid. All ot’her substrates tested, namely phosphatidyl ethanolamine, phosphatidyl Lserine, phosphatidyl inositide, phosphatidic acid, lysolecithin, sphingomyelin, cerebroside, and cardiolipin were not hydrolyzed by the purified phospholipase A. Biological activities. The lethal toxicity of the various fractions obtained during fractionat,ion of the crude venom ~vas assayed by injecting various concentrations of the fractions as previously described. The results are summarized in Table V. Purified phospholipase A, was devoid of let’hal t’oxicity. The myolytic activity of 100 pg phospholipase A was very slight. Only one small section of myolytic act,ivit’y was observed in five slides prepared from five different inject)ions. No hemorrhagic activity was observed after examining the subcutaneous area of the skins of mice inject,ed with 50 pg of pure phospholipase A. Hemolytic activity was

104

TU, TABLE TOXICITY

PASSEY,

V TEST Ko. of Dose mice (pg/mouse)b dead in 24 hrC

C&I-I

100

CM-II

l;rO

CM-III

100 30 10 100 .iO 50 10 50 10 5 3 2.3 2.0

LDso be/ mouse)

'0

50

CM-IV CM-VI CM-VII DEAR-1

DEAE-II

DEAE-III tion 1) DEAE-III tion 2)

0 0 5 0 0 .i .i”

2.8

0

5

(prepara-

3 2 100

‘2” 0” 0

(prepara-

100

0

3.2

a CM-I, II, III, IV, VI, VII are fractions from CM-cellulose column; DEAE-I, II, III are fractions from DEAE-cellulose column; fractionation procedure is described in the text. b Mice weighing 20 g were selected. c Five mice were used at each dose level. d Seven mice were used at these dose levels.

measured and expressed as HUso , the concent’ration required to hemolyze 50 % of the erythrocytes. Without lecithin, the HU60 for phospholipase A was 16 pg. In the presence of 0.1 mg beef lecit’hin, the Hub0 was 0.4 pg, indicating that hemolytic activity of phospholipase A was increased 40.fold by the addition of a small amount of lecithin. The HUso for unfractionated venom in the presence of 0.1 mg lecithin was 5 pg. DISCUSSION

A two-step purification of the enzyme phospholipase A from venom of the sea snake Laticauda semifasciata has been achieved result)ing in a preparation shown to be homogeneous by a number of criteria. The

AND

TOOM

preparat,ion appears as a single, sharp, symmetrical peak when subjected to isoelrct ric focusing, and to Sephadex gel filtration. OnI) one band can bc detected n-ith zonal electrophoresis on polyact+nte at various pH values. Also, the preparnt,ion appeared to settle as a single component on ultracentrifugation nt 59,780 rpm. The specific activity of t,ltc prep aration is about equivalent IO that of t Ile preparation of Wells and Hanahnn (7), \yhich prior to this report, exhibit cd the highest specific activity of a phospholipnse A isolated from snake venom. It has been shown that, egg phosphatid~lcholine contains predominantly saturated fatty acids csterified at1 the 1 position, and unsaturated fatty acids at thr 2 position (14). The data in Table III clearly show that, the fatty acids released by both crude venom and purified phospholipase A are predominantJy unsaturated, while those remaining esterified to lysophosphatid~~lcholine arc mainly saturated. It is thus safe to conclude that the purified phospholipase A is indeed acting on t,he 2 posit,ion of phosphatidylcholine and thus can bc called phospholipase A. The molecular weight of phospholipase Li isolated from sea,snake venom was found t,o have a molecular weight of about 11,000 bl t,wo independent’ methods. This molecular weight compares to a value of 14,500 found by Wu and Tinker (6) of a phospholipas~~ A preparation from Crotalus at~.r. This value is also similar to pancreat,ic phospholipase A which has a molecular weight of 13,800 (2.5). In contrast, the two isozymes isolated from C. adalrlanteus venom by Wells and Hanahau (7) have molecular neights of 30,000. Salach (8) found that1 the molecular weights of the nine phospholipases which hr isolated from Nuja naja venom varied from S,T,OO to 22,000. It should be pointed out’ that only one phospholipase A activity could be det’ected in the sea snake venom in this study. This was also suggested by isoelectric focusing experiments where only one activity was found and by the fractionat,ion procedure where again, only one phospholipase A \vws det)ected. A number of isoelectric points have been reported for phospholipasc A of various sources. The values vary from 5.1 to 8.7 (26,

PHOSPHOLIPASE

A FROM

SEA SNAKE

T’ENOM

10.5

27). The value of 6.6 found in t,his investigator act synergist8ically, producing hemolysis tion is in agreement with the value reported to a much greater degree than either alone for phospholipase A from N. niyricollis of 6.2 (34,35). (2X). RIaeno (29) has suggested that fib?+ REFEREXCES might be substituted for Ca*+ in activat’ing 1. CONDREA, E., DE VRIES, A., AXD M~~ER, J., phospholipase A, and Marinet,ti (18) found Biochim. Biophys.dcta84,GO (19G4). 1hat a1g2+ could act’ivate phospholipase 2. CALL~I-H.~TCH,~HD, J., .~ND THOMPSO?U’, R, A from Aykistroclon piscivorus piscivorus H. S., Riochim. Biophys. ilcta 98, 128 (1965). venom. However, it’ has generally been ac3. KURUI,, P. rZ., n-(ltltrwissenschuften 63, 84 crpted that calcium was t’he only cation (1966). 4. HABERMAXN, E., AND REIZ. K. G., Biochem. 2. \vhich could activate phospholipase A from 343, 192 (1965). snake venoms (30). The results reported in 5. MEBS, D., .~ND RAUDOX~T, II. W., Na.turthis paper on CR?+ and l\Ig*+ activation wissenschajten 54, 494 (1967). confirm the recent study by Wu and Tinker G. Wn, T. W.. AND TIXICER, I)., Biochemistry 8, (6) who found Ca2+, ISi2+, Co2+, ;\Ig”+, and 1558 (1969). Cd”+ all had an uct’ivat’ing effect . The inhibi7. WELLS, &I. A., AND IIANAHAN, D. J., Biotory effect of ZI?+ in t,his investigation is also chemistry 8,414 (1969). irl agreement with these investigators who 8. SAL.~CH, J. I., TURINI, P., HUGER, J., SE~TG, found that, Hg2+, Cu*+, Ba?+, and Zn*+ all H., TISDALE, H., AND SINGER, T. P., Bioinhibited their phospholipase A preparation them. Biophys. Res. Commun. 33,936 (1969). 9. TOOM,P. M., SQUIRE, P. G., AND Tu, A. T.. from C. afrox. Biochim. Biophys. Acta 181, 339 (1969). The substrat,e specificit,y of phospholipase 10. COREY, J. E., AND WRIGHT, E. A., Nature A isolated from the sea snake, Laticaucla London 186, 104 (1960). senrifasciafa, venom appears to be much 11. U~ATOII., NIEr;SES-

106

TU,

W., AND VAN DEENEN, L. L. M., Biophys. Acta 169, 103 (1968). HABERMANN, E., AND REIZ, K. G.,Biochem.Z. 341, 192 (1965). DAWSON, R. M. C., Biochem. J. 88,414 (1963). HABERMANN, E. A., Physiol. Chem. 297, 104 (1954). MAENO, H., MITSUHASHI, S., OKONOGI, T., HOSHI, S., AND HOMMSN, M., Jap. J. Exp. Med. 32,55 (1962). CONDREA, E., AND DE VRIES, A., Tozicon 2, 261 (1965). WIZEN,

Biochim.

26. 27. 28. 29.

30.

PASSEY,

AND

TOOM

E., AND COHEN, 8. s., J. Biol. Chem. 129,619 (1939). 32. OKUYAMA, H., AND No~rfi1.4, 8., J. Biochem. Tokyo 67, St9 (1965). 33. ALOOF-HURSCH, S., DE VRIES, A., AXD A., Biochim. Biophys. dcla 164, BERGER, 53 (1968). 34. CONDREA, E., DE VRIES, A., AND MAGER, J., Biochim. Biophys. Acta 84, 60 (1964). Pathophysiological 35. Tu, A. T., in “The Action of Neuropoisons” (Lance L. Simpson, ed.), Chap. 4. Plenum, New York, in press (1970). 31. CHARGAFF,