Toskoe . Vol. 18, pp. 675-b80.

0041-0101/80/1101-0675 9D2A0/0© Pagamnn Prtas Ltd 1980. Prinrod m lhat Hrltain

IMMUNOLOGICAL COMPARISON OF PHOSPHOLIPASES A2PRESENT IN RATTLESNAKE (GENUS CROTALUS) VENOMS

CHITRA NAIR,* B . C. NAIRt ândW. B. ELLIOTTDepartment of Biochemistry, State University of New York at Buffalo,

Buffalo, NY 14214, U.S .A.

(Accepted jor publication 18 August 1980)

C. NAIR, B. C. NAIR andW. B. ELLIOTT. Immunological comparison ofphospholipasea Az present inrattlesnake (genus Crotales) venoms . Toxicon 18, 67580, 1980.-The immunological properties ofphoapholipaseAz presentin venomsfromseven species of rattlesnakes (genus Crotales) from different~ographical distributions have been compared. Rabbit antiserum to purified PLAs of Crotalesscutulatus salvini venom was used, employing inhibition of enzymic activity and immunodiffusiontechniques. The amount ofantiserum required for 50"/inhibition ofthe PLAZ activity in thevenomsvaried from slightly more than that required for inhibition oftheactivity in thehomologous venom toa large amount (effectively zero inhibition). A particularly close relationship in the antigenic makeupwas observed between the phospholipases of C. s. salvini and C. atrox venoms as evidenced byinhibition ofenzymic activity and immunodiffusion studies. No immlmological cross reactivity wasobserved betwcen the enzymes of the subspecies C. s. salvini andC. s. scutulatus . The results suggestextensive variations in the antigenic structure of the phospholipase molecules.The findings may alsohave some taxonomic significance.

THE genus Crotalus has several species and subspecies widely distributed in Americancontinents. They vary extensively in their venom constitution and toxicity . PhospholipaseAZ(PLAZ) (phosphatide 2-acylhydrolase, EC 3.1 .1 .4) has been purified from Crotalus adaman-teeS (SATfO andHANAHAN, 1962 ; WELLS andHANAHAN, 1969), CTOtaIUS atrOX (WU and TINKER,1969 ; HACHIMORI et al ., 1971), CTOtaIeS derissus terl'ifecus (HABERMANN and RU9SAMEN,1971 ; BREITHAUPT el al., 1975) and Crotalus scutulatus salvini (NAIR et al ., 1979). PLAZisolated from various Crotalus venoms showed variations in both catalytic and molecularproperties . Very little work has been done on the antigenic properties of the enzyme.

Studies on the inhibition of PLAZ activity of various snake venoms (NAIR et a1.,1975) andinsect venoms (NAIR et al., 1976x) with antibodies to homologous and heterologous PLAZshowed varying degrees ofcross reactivity or even no cross reactivity among certain speciesofthe same family. Such differences reflect the variations in the antigenic sites of the enzymemolecule belonging to the same family. Recently MEIJER et al. (1978) studied theimmunological properties of pancreatic PLAZ and found considerable immunologicaldifferences between pig, horse, cow and sheep enzymes.

In the present study we have chosen various species and subspecies of the same genus in

" Present address : Department of Biological Sciences, Cell and Molecular Biology Division SUNY at Buffalo,Buffalo, NY 14260, U.SA.t Prescht address : Department ofOral Biology, SUNY at Buffalo, 4510 Main Street, Buffalo, NY 14226, U.S.A .

To whom all correspondence should be sent .

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order to determine the antigenic variations ofPLAZ present in the venoms ofselected closelyrelated species. Rabbit antiserum directed against purified PLAZ from C. s. salvini was usedas the source of antibody against PLAZ and the studies used both inhibition of enzymicactivity and immunodiffusion techniques.The following lyophilized venoms were purchased from Miami Serpentarium (Miami,

FL) : Crotalus scutulatus salvini (Huamantlan) lot No. CSS4TLZ, Crotalus scutulatusscutulatus (Mojave), Crotalus atrox (Western diamondback) lot No. CXA3TP, Crotalusadamanteus (Eastern diamondback) lot no. CA2-1, Crotalus horridus (Timber) lot No. CH44-12, Crotalus basiliscus (Mexican West Coast) .lot No. CBOtL and Crotalus durissus terriflcus(South American) CTZ32-12. Bovine serum albumin (fatty acid poor) was purchased fromMiles Laboratory. Pure phospholipase AZ from the venom ofC. s. salvini and rabbit antiseradirected against the purified PLAZ were prepared as previously described (NAIR et a1.,1979).Stock solutions of PLAZ and the venoms were made by dissolving the lyophilized samplesin double distilled water and the solutions were stored as small aliquots at -20°.Hydrogen ion release assay (Da HAAS et al., 1968) slightly modified by NAIR et al. (1976b)

was used to determine the PLAZ activity. Inhibition of PLAZ activity by rabbit antisera topurified PLAZ from C. s. salvini was studied as described previously (NAIR et al., 1975) .

Immunodiffusion of crude venoms of various species against anti-PLAZ serum was doneon plates prepared with 2'/ special Agar Noble (Difco) gel in 005 M sodium barbituratebuffer (pH8~2). The agar plates were placed in a humidor for 24 hr at room temperature.

Inhibition ofthe PLAZ activity ofvarious venoms by increasing the amount of antiserumagainst purified PLAZ from C. s. salvini is shown in Fig. l . PLAZ activity ofC. s . salvini and C.atrox venoms was completely inhibited by 50 Etl of the antiserum while 100 pl of antiserum

FtG . t . INHIBITION OF VENOM PLA= FROM SELECIED Crotales SPECIFS HY ANTISERUM AGAINSr PLAZFROM C. S. 801Di71i VENOMS

p C. atrox, ~ C . adaMmueus, ~ C.k . hwrides, ~ C .s. scutulatut,0 C. d. terr(ficus, C] C. baadliscus, ~C. s. salvini.

FIG. T. IMMUNODIFFUSION OF VARIOUS CRUDE CROTAL>DAE VENOMS WITH ANTISERUM (AS) AGAINSTPLAZ FROM C. s. saluini .

(1) C. s . salvini venom, (2)C. atrox venom, (3) C. s. scutula[us venom, (4) C. basüiscusvenom, (5) C. d.terrificus venom, (6) C. adamanteus venom.

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was required for total inhibition ofthe PLAZ activity of C. adamanteus venom. Onehundredmicroliters of antiserum decreased by 60~ the PLAZ activity in C. h. horridus venom and nofurther decrease was noticed on incubating with 500 pl of the antiserum. No inhibition ofPLAZ activity was observed in the case of C. basiliscus, C. s. scutulatus and C. d. terr~cusvenoms even on incubation with 500 pl of the antiserum .

ïmmunodiffusion experiments (Fig. 2) revealed that the antiserum to purified PLAZ fromC. s. salvini gave single precipitin lines with identity when crude venoms of C. s. salvini and C.atrox were tested, indicating homologous immunological reactions between the enzymemolecules of the two species. The antiserum also produced a single precipitin line with crudevenom of C. adamanteus. This band fuses with that of C. s. salvini but lacks some of thedeterminants, since the latter forms a spur . Antiserum did notgive any precipitin lines with C.basiliscus, C. s. scutulatus and C. d. terrificus. Varying concentrations ofC. h. horridus venomwas tested with the antiserum and no precipitin line was noticed (data not shown).

Conformational change of theenzyme molecule or steric hindrance of the catalytic site dueto the interaction ofthe enzyme with its antibodies maycause decrease in the enzymic activity(Aexox, 1974). A recent study (IVIEiJER et al., 1978) demonstrated that from the possiblenumber of antigenic sites of PLAZ, only three sites can simultaneously be occupied byantibody and inhibition of the micellar binding site inhibits the enzymic activity. Ourinhibition studies and immunodiffusion experiments show close identity between the PLAZmolecules of C. s. salvini and C. atrox venoms suggesting close structural relationshipbetween the antigenic sites of these two enzyme molecules . The fact that the enzyme of C.adamanteus venom was completely inhibited by the antiserum and showed immunologicalcross reactivity with lesser antigenic determinants than the enzymes from C. s. salviniand C.atrox suggests that this enzyme differs only slightly from C. s. salvini or C. atrox PLAZ in itsstructure . In the case ofC. adamanteus andC. h. horridus enzymes there is not much differencein the amount of antiserum required for 50'~ inhibition of the activity. However, 100 pl ofantiserum could inhibit the PLAZ activity ofC. adamanteus venom almost completely but noinhibition beyond 63% of PLAZ activity present in C. h. horridus venom was possible witheven 500 pl ofthe antiserum . It is possible that the venom contains two PLAZ, one ofwhich isinhibitable and the other is non-inhibitable by the antiserum, or that this venom sample is amixture oftwovenoms, one ofwhich isinhabitable andone non-inhabitable by this antiserum.Although the antiserum inhibited 63~ of the PLAZ activity in C. h. horridus venom, noprecipitin line was noticed in the immunodiffusion experiments. It is possible that theantibody recognizes only a part of the antigenic determinants of the enzyme and forms asoluble antigen-antibody complex. No immunological cross reactivity was observedbetween this antiserum against C. s. salvini PLAZ and C. basiliscus, C. s. scutulatus and C. d.terrifieus venoms.The results obtained with immunological comparison of the phospholipases AZ from

horse, pig, cowand sheep pancreas indicate variations in the antigenic determinants oftheseenzyme molecules (MeueR et al., 1978) suggesting structural alterations during molecularevolution. The results of our studies show vast differences in the immunologic activities ofphospholipase AZ present in the venoms of very closely related species . It is interesting toobserve that there is a striking difference in the immunological activities ofthe two subspeciesthat we have studied, i.e . C. s. salviniand C. s. scutulatus . The results of our studies provideimmunological evidence for structural variations in the PLAZ ofthese closely related species.The structure-function correlation of homologous proteins from closely related species cancontribute towards the understanding of evolutionary change and taxonomic status ofvarious venomous snakes.

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REFERENCES ,

ARNON, R. (1974) Immunochenistry ofenzymes. In : The Antigens, Vol. 1, pp . 109-116 (SELA, M., Ed .) . NewYorkAcademic Press.

BREITHAUPT,H., OMORI-SATOH, T. and LANG, J. (1975) Isolation and characterization of three phospholipases Afrom the crotoxin complex. Biochtm. biophys . Acts 403, 255.

DEHAAS, G. H., POSTEMA, N. M., NIEUWENHUIZEN, W. 8nd VAN DEENEN, L. L. M. (1968) PUIifiCatlOII andproperties of phoapholipase A from porcine pancreas . Biochim. biophys . Acts 199, 103.

HABERMANN, E. 8nd RUBSAMEN,K. (1971)Biochemical and pharmacological analysis ofthe so-called crotoxin . InToxins ofAnimal and Plant Origin, lst Edn., Vol. l, pp. 333-341(nE VRtes,A. and KOCFIVA, E., Eds.). NewYorkGordon & Breach .

HACHIMORI, Y, WELLS, M. A. and HANAHAN, D. J. (1971) Observations on the phospholipase A2 of Crotales atrox .Molecular weight and other properties . Biochemistry 10, 4084.

MEIJER,H., MEDUENS,M.J. M., DIJHMAN, R., SLOTaooM, A. J. and DEHAAS,G. H. (1978)Immunological studiesinpancreatic phosphoGpase AZ. J. biol. Chem . 293, 8564 .

NAiR, H. C., NAIR,C. and ELL10TT, W. B. (1975) Action of antisera against homologous and heterologous snakevenom phospholipase A~. Toxicon 13, 453.

NAIR, B. C., NAiR, C., DENNE, S., WYPYCH, J., ARBESMAN, C. E. and ELLIOTT, W. B. (1976x) Immunolopjccomparison of phospholipases A present in hymenoptera insect venons . J. Allergy clin. Immun. S8, 101.

NAIR, C., HERMANS, J., MUNJAL, D. and ELLIOTT, W. B. (19766) Temperature stability of phospholipase Aactivity-I . Bce (Apjs mellifera) venom phoapholipase AZ. Toxicon 14, 35 .

NAIR, B. C., NAIR, C. and ELLIOTT, W. B. (1979) Isolation and partial characterization ofa phospholipase A, fromthe venom of Crotales scututatus salvini. Toxicon 17, 557.

SAITO, K. and HANAHAN, D. J. (1962) A study of the purification and properties of phospholipase A of Crotalesadamanteus venom. Biochemistry 1, 521.

WELLS, M. A. 8nd HANAHAN, D. J. (1969) Studies on phospholipase A. I . Isolation and characterization of twoenzymes from Crotales adamanteus venom. Biochemistry 8, 414.

Wu, T. W. and TINKER, D. O. (1969) Phospholipase AZ from Crotales atrox venom. I. Purification and someproperties. Biochemistry 8, 1558 .