Toxtcoas, VoL 17, PP . SS7-3b9 . © Aupamon Ama Ltd. 1979. Aimed In Great Hrltain.

0041-0101/79/1101-0357102.00/0

ISOLATION AND PARTIAL CHARACTERIZATION OF A PHOSPHOLIPASE A2 FROM THE VENOM OF CROTALUS SCUTULATUS SALYINI

BALA C. N~nt, CxizztA NA>R and Wu.LaxD B. ELLIOTT Department of Biochemistry, State University of New York at Buffalo, Buffalo, NY 14214, U.S.A. (Acceptedjorpublication 19 February 1979) B~ C. Nam, C>~rtu Nem and WII.LARD B. Bi.LIO'IT. Isolation and partial characterization of a phaspholipase A, from the venom of Crotalus scutulatus salvirti . Toxicoe 17, 557-569, 1979.-A phospholipase A, (EC 3.1 .1 .4) has beon isolated and purified from the venom of Crotalusscutulatussalvürün33~yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was in the same range (30,000) whether determined by ®e1 filtration or ultracentrifugation . On polyatxylamide gel electrophoresis, in the presence of sodium dodecyl sulphate and ß-mea+captcethanol, the caryme migrated into a singlo protein band with a mobility corresponding to about 14,000 molecular weight, indicating that the native enzyme was adieter. Amino acid composition of theonzyme is reported. Two NH,-amino acid terminals were found which indicates that the dissociable monomers of the native enzyme were non-identical. Antiserum against the purified enzyme completely inhibited the phospholipaso A, activity and immunodiffusion of the antiserum against both purified phospholipase A, andcredo venom of Cratalus scutulatus salvini gave single precipitin lines indicating a lack of contaminating antigens . Tl~ csxvs Crotales has a large number of species and sub-species which differ extensively in their venom characteristics . Purification and characterization of phospholipase As (phosphatide 2-acylhydrolase, EC 3.1 .1 .4) from venoms of only a few species have been made : Crotales admnanteus (SArro and HerrAxAx, 1962 ; WIZi.i 4 and HANAxAx, 1969), Crotales atrox (Wu and Tnvx~t, 1969 ; HACLiIMORI et al., 1971) from Eastern and Western areas of the United States respectively and Crotales durissus terrifices (HAS>ï>:MAxty and RuasA~r, 1971 ; BREIT'HAUPT et al., 197 from South America. Kinetics of substrate hydrolysis of C. adamanteus (Got et al., 1971 ; W>~.ls, 1971, 1972, 1974 ; S118N et al., 197, C. atrox (CoLBS et al., 1974 ; WO1a.R and P~L.>~t-ICHIRAWA, 1974), and C. d. terrificus (RoxoLr and SCHALAMOWITZ, 1961) phospholipases A, have been studied. Recently, primary structural studies of phospholipase As from C. adamanteus (TSAO et al., 1975) and have been made . C. d. terrificus (BREITHAUPT et al., 1974 ; OMORI-SATOx et al., 197 Phospholipases A, isolated from the various species of Crotales showed variations in both catalytic and molecular properties. Phospholipase A, is an important tool for the study of metabolism and structure of phospholipids and lipid-protein interactions . Much work has been done on phospholipase As substrate requirements, metal ion activation and inhibition, kinetic parameters and amino acid composition, but very little is known about the antigenic characteristics of these enzymes. Phospholipases A, were shown to have antigenic properties by several workers (FLl3CSSNSrSIN et al., 1951 ; H~B»arnv and EL Kax>~n, 1956 ; Muic and ArDUxovlc, 1957 ; BAxx>~e et al., 1966 ; MUN7AL 8nd ELLIOTT, 1971). Comparative studies of the inhibitory effect of phospholipase antibodies on the activity of phospholipase Aa present 357

558

BALA C. NAIR, CHTTRA NAIR and WILLARD B. ELLIOTT

in a number of snake venoms (Nnm et al., 1975) and in hymenoptera insect venoms (Nnus et al., 1976) showed variations in immunological properties even among the closely related species as shown by differences in the amount of antisera required for 50 ~ inhibition of a standard amount of phospholipase Aa activity. Such differences indicate the variations in the antigenic sites of the enzyme molecule of neighboring species. Separation and purification of the antigens is the first step in the investigation of the changes in primary structure of the enzyme protein that leads to the variation in antigenic sites. The present communication describes the separation, purification, characterization and immunological properties of a phospholipase As isolated from the venom of Crotales scutulatus salvini (one of the two sub-species of Crotales scutulatus) which is known as the Huamanthan rattlesnake and is found in South Central Mexico . Materials

MATERIALS AND METHODS

Lyophilized venom of C. s . salvini (Lot No . CSS4TL27 was obtained from Miami Serpentarium (Miami, FL). Bio-Gel P-10, P-60 and P-150, used for gel filtration, and DERE cellulose (Cellex D), used for ion exchange separations, were purchased from BioRad Laboratories (Richmond, CA). Sulfopropyl (SP) Sephadex A-25 from Pharmacia (Piscataway, Nn was also used for ion exchange separation . Aldolase (145,000), ovalbumin (45,000), chymotrypsinogen A (25,000), myoglobin 17,800), ribonuclease A (13,500) and cytochrome c (12,400) from Mann Laboratory, were used as standard proteins for molecular weight determinations . Protein staining was done with Coomassie Brilliant Blue G-250 (Serve Feinbiochemicals, Heidelberg). Spocial Agar-Noble, used for immunodiffusion, was purchased from Difco Laboratories, Detroit, MI. Methods Measurements ofphospholipase A, activity. The activity measurement was carried out in a microreaction

vessel by automatic titration of the fatty acid liberated from an egg yolk substrate with 0"01 N NaOH, using an automatic titrimeter (Radiometer, Copenhagen, pH-stet model TTll/SBR2/ABU12/TTA31) at 37°C and pH 8"0. The method of D$ Hnas et al. (1968) slightly modified (None et al., 1975) was used. One unit of phospholipase A, activity released 1 umole of fatty acid/min . Purtfrcatton procedure . All purification steps except ultrafiltration and dialysis were carried out at room temp . Dry venom (250 mg) was suspended in 3 ml of 002 M ammonium formate buffer (pH 4" 5), containing 0001 M EDTA and centrifuged at 2000 g for 10 min. The slightly turbid supernatant (sol . n, containing all the phospholipase A, activity, was chromatographed on Bio-Gel P-10, P-60 and P-150 in tandem columns. Gel filtration . Bio-Gel P-10, P-60 and P-150 were allowed to swell in 002 M ammonium formats buffer containing 0001 M EDTA (pH 4"5) . Each of the deserated, swollen gels was packed into a glass column (Pharmacia) 2"5 x 100 cm . The first of the three tandem columns (P-10) had descending flow andthe second (P-60) and the third (P-150) had ascending flow . The columns were equilibrated overnight and the flow rate adjusted to 15 ml/hr. Solution I was applied to the column and was eluted with the same buffer used to pack the columns. The transmission of the e®uent was monitored (LKB UVCord In at 280 nm and the effluent was collected in 4"0 ml fractions (flow rate 15 ml/hr). Fractions with phospholipase A, activity were pooled and lyophilized. The lyophilized solid was dissolved in 10 ml of double-distilled water and then dialyzed against 001 M phosphate buffer (pH 7~4) at 4°C (the dialysis tube was boiled in O1 M EDTA for 15 min and washed thoroughly with double-distilled water before use) . The dialyzed solution (sol . In was subjected to ion exchange separation on DEAE cellulose. DEAE Cellulose chromatography . Cellex D (0 "96 meq/g) was precycled as suggested by themanufacturers, equilibrated with 001 M sodium phosphate buffer (pH 7"4) and packed into a 2 x 20 cm column . The column was equilibrated overnight in 001 sodium phosphate buffer, maintaining the flow rate at 20 ml/hr. Solution II was applied to the column and the column was eluted with 200 ml of 0"01 M phosphate buffer (pH 7~4) followed by a linear gradient formed from 200 ml of starting buffer and 200 ml of 02 M NaCI in starting buffer. The eluate was collected in 4"0 ml fractions. Fractions with phospholipase A, activity were pooled and wncentrated to 10 ml at 4°C, using an ultrafiltration cell (Amicon) with a U.M .-10 membrane. The concentrated solution was dialyzed against 0"01 M ammonium formats buffer (sol. IIn and chromatographed on SP Sephadea A-25 . SP Sephadex chromatography. SP Sephadex A-25 was permitted to swell in 001 M ammonium formats buffer (pH 4"5) and then packed into a 2 x 20 cm column . T'he column was equilibrated overnight with the same buffer at a flow rate of 15 ml/hr. Solution III was applied and thecolumn was eluted with the same buffer and the eluate was wllected in 4"0 ml fractions. Fractions with phospholipase A, were pooled and concentrated by ultrafiltration as described above. Ammonium formatswas removed by three cycles of concentration in an ultrafiltration cell with U.M :10 membrane. The salt-free concentrated solution (sol. I~ was lyophilized.

Crotalus acutulatus salvi>!i Phospholipase A,

559

Polyacrylamide gel electrophoresis. Tho proteins from each step of purification were eleclrophoresed in 7~5~ polyacrylamide gel using the buffer system of Cuxxt: (1964) . Electrophoresis was carried out at room temperature, applying a constant current of 2 mA/running tube. Proteins were stained with Coomassie Brilliant Blue G-250 (DiEZEL et al., 1972). Protein estimation . Protein concentration was estimated by the method of LowxY et al. (1951) using bovine serum albumin as the standard protein. Molecular weight determinations-$el jrltration. The columns used for gel filtration were calibrated with molecular weight markers; aldolase, ovalbumin, chymotrypsinogen and cytochrome c. Molecular weights were estimated by the use of the AxnaEws' method (1964) . Ultracentrifugation . The sedimentation equilibrium experiment was made in a Spinco Model E ultracentrifuge equipped with Rayleigh interference optics . The experiment was performed using a 3 mm column in a double-sector cell (30 mm optical path at 20° and 13,410 rev/min for 30 hr. The protein solution (4 mg/ml) was made in 0~1 M phosphate buffer (pH 7~4). The changes in concentration throughout the cell were followed by means of a fine wire mesh interposed in front of the photographic plate, and taking photographs at suitable intervals during the course of the experiment (DexaccFmv, 1969). Thus, the fringe displacement could be followed from the start of the experiment and the analysis of the plot made with precision. The molecular weight was determined using Svedberg's equation (with the usual symbolism) : M

_

RT (1 -9p)

dlnc dr'

Apparent partial specific volume 9 was assumed to be 0"74. Sodium dodecyl sulphate polyacrylamide gel electrophoresis. The method of Wssat and OsHOxN (1969) was followed for gel electrophoresis in the presence of sodium dodecyl sulphate, using standard molecular weight markers (ovalbumin, 45,000 ; chymotrypsinogen A, 25,000 ; myoglobin, 17,800 ; ribonuclease A, 13,500 ; and cytochrome c, 12,400) in 10~ polyacrylamide gel containing 0~1 ~ sodium dodecyl sulphate and 0~1 % ß-mercaptcethanol. The standard proteins and purified phospholipase A, were incubated for 2 hr at 37 °C in the presence of 0~1 ~ sodium dodecyl sulphate and 0~1 % ß-mereaptoethanol. The electrophoresis was done at 8~0 mA/gel for several hours. Amino acid analysis. Amino acid analyses of the protein (1 mg) were carried out by the method described by MOORE and S~ (1963) on a Beckman analyzer (Model 120 B). The NH,-terminal amino acid analysis was performed using dansyl chloride as described by Gxwv and Haxzt,ev (1963). Three-tenths mg of the sample was treated with approximately 20 ~1 of 0~1 M Na °CO, solution and dried at room temperature on P,O, . About 15 ul of 0~2 M NaHCO, and 25 ~1 of dansyl chloride (2 mg/0~7 ml) in acetone were added to the sample, and the mixture was allowed to stand for 15 min at about 50°C. The above solution was dried in a desiccator and heated under vacuum. The dansylated protein was hydrolyzed in 100 ul of 6 N redistilled HCl by holding for 6-8 hr at 110°C in an evacuated sealed tube . The sample was dried and extracted in a formic acid and water solution . Standards wero applied and run in butanolheptane-acetic acid solvent. Preparation ojantiserum. Fifty ug of purified phospholipase A, were emulsified in 0~3 ml of Freund's complete adjuvant and subcutaneous injections were given at multiple sites on a rabbit . One week later, a booster injection was given with 50 pg of phospholipase A, emulsified in 0~3 ml of incomplete Freund's adjuvant. The rabbit was bled through the artery of the ear one week after the booster injection. Increased titre of the antiserum was obtained after a third injection with the same amount of phospholipase A, used in the previous injection. Imnrunodiffusion . Diffusion of purified phospholipase A, or crude venom of C. s. salvini against antiphospholipase A, serum (rabbit), on plates prepared with 2~ special Agar-Noble gel in 005 M sodium barbiturate buffer (pH 8~2), was done for 24 hr in a humidor. Inhibition study. Varying amounts of antiserum were added to 50 pl aliquots of purified phospholipase A, containing 2~3 units of activity and double-distilled water was added to the mixture in order to keep the final volume of 200 ul. After the mixtures were incubated for 30 min at 37°C and overnight at 4°C, the tubes were centrifuged at 2000 g for 10 min. One hundred kl aliquots of the supernatant solution were assayed for phospholipase A, activity . RESULTS

Results of the step-wise purification from two experiments using the same lot of venom are given in Table 1 . The elution profile of one of the gel filtrations is shown in Fig. 1 . A sample of 250 mg could be successfully chromatographed at one time. Phospholipase As activity was found in only one peak . Disc gel electrophoresis at pH 8~6 of the concentrated pooled phospholipase As fractions from gel filtration showed at least 11 bands staining strongly for protein (Fig. 4A).

360

BALA C. NAIR, CHITRA NA1R and WILLARD B. ELLIOTT T.~LE 1. PURIFICATION OF VENOM PHOSPHOLIPA3E FROM Crotdrrs scutulatus salvini Purification step Credo venom Supernatant Gel filtration (tandem column) DEAF Cellulose SP Sephadex

Protein (m~ 250 250 235 233 529 535 835 870 510 3 60

Total activity in units'

Specific activity in unitst

24,205 24,203 20,000 16,832 10,873 11,880 7500 8736

103 103 378 313 1310 1363 1470 1560

Yield

31~ 36

'klnole of fatty acid released per min. tulnole of fatty acid released per mg of protein per min. Experimental conditions are described in the text . Data are from two experiments on tho same lot of venom.

i

E

O m N

2.0 L5 L0 0.3

FIO. 1. PURIFICATION OF PIi08PHOLIPA~ A~ FROM CRUDE VENOM HY OEL FILTRATION ON TIiRHE COLUMNS, CONTAINIIVO BIO-GEL P-10, P-6Q AND P-130, RESFECrIVELY, CONNECT'® IN sERIPB . Venom (233 m~ applied, followod by 002 Mammonium formate buffer (NH, OH-HCOOH) (pH 4~5), containing 0001 M EDTA . Fractions of 4-0 ml were collected at a flow rate of 15 ml/hr.

The elution profile of the ion exchange separation on DEAF cellulose (Fig. 2) showed only one peak with phospholipase A, activity. The pooled active fractions had a specific activity about 13 times that of the original venom and showed only two strong protein bands (Fig. 4B), one dark and the other very faint, moving very close to each other. Chromatography on SP Sephadex (Fig. 3) separates these two components, increasing the specific activity of the pooled active fractions to about 15 times that of the crude venom. Disc gel electrophoresia of the pooled fractions from SP Sephadex chromatography showed only one band staining for protein (Fig. 4C). The maximum yield obtained was 36 ~ of the total phospholipase As activity present in the crude venom.

Crotales serrtulates saJvJnt Phospholipase A,

561

o .s É O m N

0.4

L0

~, 200

E

w

0.3

ô 0.2

-

Fraction No .

FIa. 2. PURIFICATION OF PHOBPHOLIPASE A, ON DEAE CELLULOSE (CELLEX D) . The cleats from gel filtration columns containing 535 mg of protein was applied. The column was eluted with 120 ml of 001 M sodium phosphate buffer (pH 7~4) and then with a linear gradient formed from 200 ml of 001 M starting buffer and 200 ml of 0~2 M NaCI in starting buffer. Another gradient formed from 100 ml of 0~2 M NaCI in 001 M sodium phosphate buffer and 100 ml of 0~5 M NaC7 in 0~5 M phosphate buffer was applied. Fractions of 4~0 ml were collected. Solid line indicates NaCI concentration.

Lo

E

0 m N 0 u

0 .5

ô a a Q

10

20

30

Fraction No.

FIa. 3. FINAL PURIFICATION OF PHOSPHOLiPASE Af ON SP-SEPHADEX (G25). Protein from DEAE column (8~7 mg) was applied followed by elution with 001 M ammonium formate buffer (pH 4~~. Fractions of 4~0 ml each were collected at a flow rate of 15 ml/hr.

Q

5152

BALA C. NAl1t, CHIT'RA NAIR and WILLARD B. ELLIOTT

Molecular weight determinations

By the use of gel filtration, the molecular weight of phospholipase Aa was estimated at 32,000 (Fig. 5). The sedimentation equilibrium measurements at 20°C in O~l M phosphate buffer (pH 7~4) gave a molecular weight of 30,000, assuming a v of 074, for the single

Aldolose

a O 9 x 8 7 t 6 5 3 ô 4 ûu 3

Ovolbumln Phosphlipase A Chymotrypsinogen A

ô

2 Cytochrome C L

500

I

600

~

L-

700

800

Elution volume,

I

900

ml

FIG. S. MOIE(ÛLAR WEIGHT DETERMINATION OF PHOSPHOLIPASE A~ BY OEL FILTRATION . Aldolase (158,000), ovalbumin (45,000), chymotrypsinogen A (25,000) and cytochrome c (13,000) were used as molecular weight markers on the gel filtration columns.

4

FIa.

az .o

rz G. MOLECULAR WEIGHT DETERMIIVATION OF PHOSPHOLIPASE A! FROM SEDIMENTA270N EQUII.IHRIUM MHASUREI~NTS AT 20°C IN 0~1 M PHOSPHATe BUFFER (pH 7~4).

From the same set of data, molecular weights versus protein concentration are plotted in the inset.

563

FIG. 4. POLYACRYLAM(DH GEL ELECTROPHORESIS OF THE POOLED ACTIVE FRACTION FROM (A) OEL FII,TRATION, (B) DEAF SEPARATION AND (C) SP SEPFIADEX COLiJMN.

The gel concentration was 7~5~. Tris-glycine buffer (003 M) (pH 8~~ was used in the buffer chambers . Electrophoresis was done on 4 x 75 mm gels for (A) 60 min, (H) and (G) 90 min each . In (G) 25 pg of protein were applied.

FIG . H. IMMUNODIFFUSION OF PURIFIED PHOSPHOLIPASE A~ AND CRUDE VENOMS OF C. S. SRIVlR1 AGAINST ANTIPHOSPHOLIPASE A s (C. S . SR~V~IIl) SERUM (RABBIT) AT ROOM TEMPERATURE,

lmmunodiffusion plates were prepared with 2~ special Agar-Noble in 005 M sodium barbiturate buffer (pH 8-2). The center well contained 50 ul of antiserum and the peripheral wells contained 10 ug of crude venom and 0~5 ug of purified phospholipase A, in alternating wells, starting with purified phospholipase As at the top well,

Crotales scutulatus salvlnl Phospholipase A,

S68

component present (Fig. 6). A plot of molecular weight versus protein concentration (inset Fig. ~ showed no change in the molecular weight with concentration . Sodium dodecyl sulphate electrophoresis of the enzyme showed a single band with 4,000 as 'molecular weight (Fig. ~.

a ô_ x

s a 6

s

`" Ovalbumin

L P Q .L ô

3

" Ghymotrypsinogen A

m

ô

2

I

I

0.2

04

I

\ Myoglobin -Phospholipase A 7 " Ribonuc!ease A ~"`Gytochrome C

O.G

I

08

I

LU

raowvty FIQ.

7.

MOLECULAR WIIOHT DETERMINATION OF PHOSPHOLIPASE A" BY SODIUM DODECYL SULPHATE POLYACRYLAMII)E OEL ELECTROPHORESIS.

Ovalbumin (45,000), chymotrypsinogen A (25,000), myoglobin (17,800), ribonuclease A (13,800) and cytochrome c (12,400) were used as molecular weight markers. Sodium phosphate buffer (005 M, pH 7~0), containing 0~1 ~ sodium dodecyl sulphate, was used in the buffer chambers . Gel conocntration was 10~. Electrophoresis was done for 7 hr at 8~0 mA~gel . Ay Crotales seefinales salvlni Number of residues Nearest whole Amino acid found integer Lya 1231 12 His 382 4 Arg 2721 27 Asp 2609 26 Thr 1083 11 Ser 1248 13 Glu 2814 28 Pro 1620 16 Gly 2324 23 Ala 1373 14 Half-Cys 2274 23 Val 617 6 Met 1~99 2 Ire 1174 12 Leu 1303 13 Tyr 1472 1S Phe 809 8 Trp N.D . Total 253 N.D. = Not determined . Values are expressed as moles of amino acid per mole of protein.

TABLE

2.

AMINO ACID DOMPOSITION OF VENOM PHOSPHOLIPASE of

566

BALA C. NAIR, CH[TRA NAIR and WILLARD B. ELLIOTT

Amino acid composition The amino acid analysis data (Table 2) shows 253 amino acid residues. NHm-terminal analysis data indicated two amino-terminal residues, Lys and Ser. E~ect ofpH The phospholipase As gave a sharp pH optimum at pH 8~0 with dispersed egg yolk as substrate.

Immunological studies The antiserum against purified phospholipase A$ prepared in a rabbit gave a single precipitin line, with identity, when diffused against both crude venom and purified phospholipase A9 (Fig. 8). The antiserum blocked phospholipase A$ activity and the titration curve (Fig. 9) shows that 26 lt1 of antiserum are required for 50 % inhibition of 2~3 units of homologous phospholipase A~ activity.

0

2o ao Aatlarum, ~l

so

FICA. 9 . SuPPRFSSZON OF PHOSPHOLIPASS A, AGTMTY BY ANTiPHO3PHOLIPA3E A, SHRUM . Two and three-tenths units of phospholipase A, activity were incubatedwith antiserum for 30 min at 37°C. Non-precipitated phospholipase A, activity was assayed as described by N.~e et al. (197 .

DISCUSSION

Although the isolation procedure of phospholipase As from C. s. salvini is similar to the previous procedure developed for the purification of the enzyme for venoms of C. admnanteus (WELLS and HANAHAN, 1969) and C. atrox (HACFIIMORI et al., 1971), the percentage recovery of phospholipase A, activity is higher than the yield reported for the venom of C. atrox (Hnc~oxi et al., 1971), but lower than the yield reported for C. adamanteus venom (WELLS and HANAHAN, 1969), Phospholipases isolated from C. s. salvini, C. atrox, a and ß forms of phospholipase As from C. admnanteus showed specific activities of 1500, 1500 (Hnct~oxi et al., 1971), 1415 and 1425 (Wlzr .i c and HANAHAN, 1969), respectively . No contaminating proteins were detectable in the purified phospholipase As by disc gel electrophoresis using 25 ltg of sample, sodium dodecyl sulphate polyacrylamide electro-

Crotdrrs scr~trdatua aatvtrd Phospholipaso A,

567

phoresis or by ultracentrifugal analysis . The antiserum against purified phospholipase As almost completely precipitated the enzyme activity . A single precipitation line, with identity with that due to purified phospholipase AE, when the antiserum against the phospholipase was diffused against the crude venom indicated that the isolated phospholipase As contained only one antigen capable of stimulating antibody formation . This antiserum crossreacts with phospholipase A, present in the venoms of C. atrox, C. adamanteus and C. horridus horridus but not with phospholipase A, present in the venoms of C. scutulatr~s scutulatus, C. d. terrificus or C. basiliscus. Details of this investigation will be published elsewhere . Although WEt.IS and HANAHAN (1969) have shown the existence of two molecular species of phospholipase As in the venom of C. adamanteus, the enzymes isolated from all the three closely-related species exist as differs in the native form. The molecular weight of phospholipase A, from the venom of C. s. salvini is estimated to be about 30,000 by gel filtration and sedimentation equilibrium measurements . However, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and ß-mercaptcethanol indicated the presence of a single band with a mobility corresponding to about 14,000 in molecular weight . These findings indicate that the enzyme is dissociated into monomers under denaturing conditions. Although WELIS and HANAI-IAN (1969) could not detect any NH,terminal amino acid in either type of phospholipase A$ isolated from C. adamanteus, Tsno et al. (197 have reported that the subunits in phospholipase AE dieter in a form are identical . The results of NH$-terminal amino acid analysis of the venom phospholipase A$ from C. s. salvini show two NH,-terminal amino acids, Lys and Ser, which strongly indicate that the two peptides of the native enzyme are non-identical . Phospholipase A isolated from crotoxin complex, the neurotoxin from C. d. terr~cus, has been shown to consist of a single peptide chain with 15,800 molecular weight intramolecularly crosslinked by eight disulphide bridges (BRSIT'HALTPT et al., 1974) . The data presented indicate that the venom phospholipase As of C. scutulatus salvini has two, probably non-identical, subunits of molecular weights of about 15,000. The native phospholipase A$ molecule does not associate or dissociate under the conditions utilized for gel permeation or ion exchange chromatography, disc gel electrophoresis or ultracentrifugation . The soluble portion of the crude venom appears to contain about 6 phospholipase A, (calculated on the assumption that no inhibitor is present in the crude venom and that no activator is lost during purification which is not supplied in the phospholipase A$ assay medium) . The molecular weight, dimeric nature and optimum temperature for the activity of this phospholipaseA s areveryclose to those reported by others for venom phospholipase As from other species of Crotales (WELLS and Hnxnxnx, 1969; HACHIMORI et al., 1971). The amino acid analysis differs by 37 amino acid residues from that of C. admnanteus (HEINRIKSON et al., 1977) and 27 amino acid residues from that of C. atrox. The findings ofboth Lys and Ser as HN,-terminal amino acids is markedly different from the finding of serine as the NH,-terminal amino acid on both dieters of phospholipase A, from C. adamanteus by sequencing (HEIN1tIRSON et al., 1977) . The presence of two NH,terminal amino acids indicates that the two subunits are not identical, that the NH,terminal amino acid varies with the individual snake or that two phospholipases A$, each with identical subunits but with different HN,-terminal amino acids, are produced by each snake . Clarification of this point will require venom collected from individual snakes or fangs of snakes. Ackrmwtedgement.~C. Nnrn was a United Health Foundation of Erie County fellow. This research was supported in part by USPHS grant GMl)6241 . We thank H. JORDAN for the ultracentrifuge studies and JvnY Wor t~ for the line drawings.

56 8

BALA C. NAIR, CHTTRA NAIR and WILLARD B. ELLIOTT REFERENCES

Axnxgws, P. (1964) Estimation of molecular weights of proteins by Sephadex gel filtration . Biochem. J. 91, 222. Bnxx~e, S. A., Mgr_, A. W., WALT'ON, K. W. and WESTOrr, P. D. (1966) Separation and isolation of hyaluronidase and phospholipase components of bee venom and investigation of bee venom-human serum interactions. Clin. chim . Acts 13, 582. Bxsrrxaurr, H., Ruaca~x, K. and HnsateMatvta, E. (1974) Biochemistry and pharmacology of the crotoxin complex. Biochemical analysis of crotapotin and the basic Crotalus phospholipase A. Eur. J. Biochem. 49, 333. BF.EITHAUPT, H., O~oiu-Snrox, T. and Lnxa, J. (1975) Isolation and characterization of three phospholipases A from the crotoxin complex. Biochim. biophys. Acts 403, 255. Ciax~, J. T. (1964) Simplified `disc' (polyacrylamide gel) electrophoresis . Ann. N.Y. Acad. Sci. 121, 428. COLE9, E., Mcùwauv, D. L. and RAPPORT, M. M. (1974) The activity of pure phospholipase A, from Crotalus atrox venom on myelin and on pure phospholipids . Biochim. biophys . Acts 337, 68 . D$Iiaas, G. H., PosrEeeA, N. M., Nix, W., VAx DEeiv>?tv, L. L. M. (1968) Purification and properties of phospholipase A from porcine pancreas. Biochim. biophys. Acts 159,103. Dettactmv, M. (1969) Modification of the model E analytical ultracentrifuge for sedimentation equilibrium study. Analyt. Biochem. 28, 385. DiBZZEL, W., KOPPIIt9CC~AGER, G. and HaPasArrx, E. (1972) An improved procedure for protein staining in polyacrylamide Bols with a new type of Coomassie brilliant blue . Analyt. Biochem. 48, 617. F~c~vsr$u~r, A., BERG, G., GenreR, J. and $CHOHIG, S. (1951) l;Jber die dehydrasen hemmung durch schlangengifte and die inaktivierung den dehydrasen hemmenden prinzips durch antitoxische sera. Arch . exp. Path . Pharmak. 213, 265. GoERxE, J., DE GAR, J. and BotvsEtv, P. P. M. (1971) Silica gel stimulates the hydrolysis of lecithin by phospholipase A. Biochim. biophys. Acts 248, 245. GRAY, W. R. and HaRrt.Er, B. S. (1963) The structure of a chymotryptic peptide fmm Pseudomonas cytochrome c-551. Biochem. J. 89, 379 . HAHERMANN, E. and EL KAREMI, M. M. A. (1956) Antibody formation by protein components of bee venom. Nature, Land. 178, 1349 . HAHERMANN, E. and RuaseMex, K. (1971) In : Toxins ojAnimal andPlant Origin, (UEVRIF3, A. and KocxvA, E., Eds.), 1st ed., Vol. 1, pp . 333-341 . New York : Gordon and Breach . HACFDaiORI, Y., WEUS, M. A. and HANaxArr, D. J. (1971) Observations on the phospholipase A, of Crotalus anox . Molecular weight and other properties. Biochemistry 10, 4084. HEtrvRixsox, R L., KRUECiER, E. T. and KEnK, P. S. (1977) Amino acid sequence of phospholipase A,-a. from the venom of Crotalus adamanteus. J. biol. Chem . 2-52, 4913 . LowRY, O. H., RO3EHROUGH, N. J., FARR, A. L. and RaNOArs., R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem . 193, 265. MooRE, S. and SzEert, W. H. (1963) Chromatographic determination of amino acids by the use of automatic recording equipments . Meth . Enzym. 6, 819. Mtnc, N. and Amugon+ic, J. (1957) The antiphosphotidase A activity in relation to the antitoxin properties of Vipers ammodytes antivenin. Arhiv. Jug. rada. 8, 89 . M[7NJAI, D. and Er.e toz-r, W. B. (1971) Studies of antigenic fractions in honey bee (Apts mellijera) venom . Toxicon 9, 229. None, B. C., NAne, C, and Euxo~rr, W. B. (1975) Action of antisera against homologous and heterologous snake venom phospholipase A, . Toxicon 13, 453. Nam, B. C., NAUe, C., DExtvE, S., WYPYCH, J., ARHE3MAN, C. E. and ELLIOTT, W. B. (1976) Immunologic comparison of phospholipases A present in hymenoptera insect venoms . J. Allergy clin. Immun. 58, 101. OMORI-SAroti, T., LANs, J., BaErrFtauPr, H. and HAHERMANN, E. (1975) Partial amino acid sequence of the basic Crotalus phospholipase A. Toxicon 13, 69. Roxotr, O. and SCFII.AMOWITZ, M. (1961) Studies of the use of dihexanoyl lecithin and other lecithins as substrates for phospholipase A with addendum on aspects of micelle properties of dihexanoyl lecithin . Archs Biochem. Biophys 94, 364. SArro, K. and HANaxAN, D. J. (1962) A study of the purification and properties of phospholipase A of Crotalus adamanteus venom. Biochemistry 1, 5~1 . St-mN, B. W., TsAO, F. H. C., Law, J. H. and KEZDY, F. J. (1975) Kinetic study of hydrolysis of lecithin mono-layers by Crotalus adamanteus a-phospholipase A, monomer dimer equilibrium. J. Am . Chem . Soc. 97, 1205 . Tseo, F. H. C., KEUK, P. S. and HENRtcxsoN, R. L. (1975) Crotalus adamanteus phospholipase A,-a, subunit structure with terminal sequence and homology with other phospholipases. Archs Biochem. Biophys. 167, 706. WEBER, K. and OSBORN, M. (1969) The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis. J. biol. Chem . 244, 4406.

Crotales scutulatus salvfni Phospholipase A,

569

W~.i.s, M. A. (1971) Evidence for O-Aryl cleavage during hydrolysis of 1,2-diacyl-Sn-glycerol-3-phosphoryl choline by the phospholipase A, of Crotales adamanteus venom. Biochim. blophys. Acta 248, 80. Wsus, M. A. (1972) A kinetic study of the phospholipase A, (Crotales adamanteus) catalyzed hydrolysis of 1,2-dibutyryl sn-glycerol-3 phosphoryl choline. Biochemistry 11, 1030. Wens, M. A. (1974) A kinetic study of the phospholipase A, (Crotales adamanteus) catalyzed hydrolysis of 1,2-dibutyryl-Sn-glycero-3-phosphoryl choline. Biochemistry 13, 2248 . Weus, M. A. and HANAHAN, D. J. (1969) Studies on phospholipase A.-I. Isolation and characterization of two enzymes from Crotales adamanteus venom. Biochemistry 8, 414. Woet.x, H, find PEILER-ICHIKAWA, K. (1974) The action of phospholipase A, purified from Crotales atrox venom on specifically labeled 2-aryl-l-alk-ll~nyl and 2-aryl-I-akyl-Sn-glycero-phosphoryl choline. FEES Lett. 45, 75 . Wv, T. W. and TnvxEx, D. O. (1969) Phospholipase A, from Crotales atrox venom.-I. Purification and some properties. Biochemistry 8, 1558 .

Isolation and partial characterization of a phospholipase A2 from the venom of Crotalus scutulatus salvini.

Toxtcoas, VoL 17, PP . SS7-3b9 . © Aupamon Ama Ltd. 1979. Aimed In Great Hrltain. 0041-0101/79/1101-0357102.00/0 ISOLATION AND PARTIAL CHARACTERIZAT...
NAN Sizes 0 Downloads 0 Views