Phospholipase A2 activity of long-chain cardiotoxins in the venom of the banded krait (Bungarus fasciatus)

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To~ccn, Vd. 21, No . 1, pp . 16-165, 1983 . Printedin arat Brhain .







WEN-CHAN(3 CxANa,l' 2





Lol. z


Inatitute of Biolokal Chemistry, Academia Simca and

Institute of Biochemical S(9ellee8, National Taiwan University, Taipei, Taiwan, Republic of China

(Accepted forpublication 19 May1982)

0041-0101/giA10163-Q3 fOß.OU"0

A1983 Perpmoo PtewLbd .

V1+.-C. Cr~xc, M.-L. Lga and T.-B. Lo. l'hospholipase A


activity of long~hain cardioto~tina in the venom of the banded krait (Bungarus faaciatua). Toxionn 21, 163-165, 1983.-Re-investigation of the long-chain cardioto~na fromBungasus fasciarusvenom reveals that they are weakphospholipases oftheA


type. The specâflc activities (units/mg) toward egglecithin are 0.42,

1.65,and0.29for the long-chain cardiotoxios V-2,V-3and VI, respectively .


toxic components (V-2, V-3andVI) isolated from thevenomof


(Miami Serpentarium Laboratories, U.S.A.) (Lu andLo,1974) have been characterized

as "cardiotoxic" as they cause contractors in a chicken biventer cervical muscle assay


SllJwu et al.,

1975). Later, their amino acid sequences were elucidated (Lu and Lo,

1978,1981). It wassurprising that they showed about 30% sequence homology with cobra

phospholipase AZ (phosphatidate 2-acylhydrolase, EC 3.1.1 .4) but did not exhibit

phosphoGpase AZ activity. Moreover, both their circular dichroic spectra and chemical

properties, such as molecular weights and amino acid compositions, were similar to that

ofphospholipase AZ and totally unrelated to typical cobra cardiotoxins (LuandLo,1981).

These facts suggested that these toxins might belong to thephospholipase A2class. In the

previous assays for phospholipase A2 activity the samples were tested at nanogram levels

and the results were negative. Recently the assays were repeated at higher doses and it

soon became clear that these toxins exhibited phospholipase A activity at high doses

(Table 1) . It is clear from Table 1 that all three cardiotoxins show significant activity,

although it is lowas compared with cobra phospholipase A2. Ourcardiotoxin preparations

have been purified and shown to be homogeneous by disc electrophoresis (pH4.3, 7.5%

gels). No extra bands were observed at doses up to 400pg. Moreover, by extracting the

proteinfrom gel slices after electrophoresis, we could demonstratephospholipase

Aactiv-ity exactly at the position corresponding to that of the protein band stained with amino

black. This was true for all three cardiotoxins. It seems reasonable to conclude that the

enzyme activity is intrinsic to these protein toxins and did not originate from other

contaminating phospholipases A which, if present, should have been removed during

extensive purification procedures and electrophoresis. Furthermore, it has to be pointed

outthat no otherfractions fromthevenomof


were active in the enzyme

assay and it was impossible for our preparations to be contaminated with more active

phospholipases A.

In order to confirm the enzyme activity on the one hand and to elucidate the site of


164 Short Communications

hydrolysis on the other, the hydrolysis products produced by the action of V-3 on egg lecithin were subjected to nuclear magnetic resonance studies as previously described. (C~wlvG andIro, 1975). The NMR spectra of lecithin and the lysolecithin and fatty acids

obtained by the action of V-3 are shown in Fig.1. It can be seen that the signals around S = 5.36 ppm (or z = 4.64ppm), which aredueto protons on adouble bond in thelecithin molecule (CHAPIIlAN and MoluusoN,1966), are missing in the spectrum of lysolecithin, but are present in the spectrum of the released fatty acids. This means that unsaturated fatty aryl groups have been completely hydrolyzed to the free fatty acids. It is well established that the unsaturated fatty aryl groups are esterified mainly at the 2-position of the glycerol moiety (IIANAHAN et al., 1960). Therefore, our results indicate that V-3 attacks the ester bond at the 2-position and is a phospholipase A2.

It has been reported that the histidine and aspartic acid residues at positions 47 and 48 in the amino acid sequence of snake venom phospholipases AZare involved in the active


The solvent used was CDA3. Chemical shifts are expressed on the 8 scale. The spectra were obtained on a JEOL FT-100 NMR spectrometer . Thelysolecithin andfatty acidsused in this study were prepared by inwbating0.5 mg of V-3 in 100Nl of 25 mM CaC12 with 1 g of egglecithin in 10 ml of diethylether for 16 hr at 37°C . Theprecipitated lysolecithin was recoveredby centrifugation and purified by repeated precipitation from methanolic solution with ether. The fatty acids remained in the etherphaseandwere extracted into0.1 MNaZCO,. On acidificationthey wenextractedwith ether. These hydrolysis products (overall yield more than 7046) gave single spots in thin"layer


Short Communications 165

'Tlte designation for these long~hain cardiotoxi~ are those of the previous report (Lu and Lo, 1981). tone unit is the enzyme activity which will please 1 wokof fatty acid in 1 min.

$This typical enzyme Preparation is included for comparison .

The enzyme activity wasmeasured by the titrimetric method in an automatic recordingpH-slat.The substrate solution was similar to that of Sraoxo aal. (1976), consisting of the following: 5mM egg-yolk lecithin; 5 mM sodium deoxycholate; 25 mM CaCh; 0.1 MNaQ; 0.1 mM EDTA. The pH was adjusted to 7.5 before use. For each away, 4 ml of the substrate solution was used and the rate ofconsumption of 2mM NaOH was measured at 37°C.



et a1.,1977 ; Ros>:R~rs et a1.,1977) . For the long-chain cardiotoxins reported

in the present paper, the histidine and aspartic acid residues are transposed to positions

39 and 4A, respectively (Lu and Lo, 1981), and this might be the reason for the drastic

decrease in enzyme activity.


Cxwxa, W.C. and Lo, T.B. (1975) The diûerentiation of phospholipases A, and AZ bY nuclear magneticresonance spectroscopy. J.

Chin. biodhem . Soc. 4,43.

Ct~raux, D. andMontrsoH, A. (1966) Physical studiesofphospholipids, IV. High resolution nuclear magnetic resonance spectra of phospholipida and related substances. J.biol. Cran. 241,5044.

Hwxwxru~c, D.J., Baoct®cao~, H. and BARYpIi, E.J. (1960) The site of attack ofphospholipase (kcithinase) A on lecithin: a re-evaluation. Position offatty acids on lecithins and triglyoerides. l.biol. Chan. 235,1917. Lna Smwu, S.Y., Hur+xa, M.C. and LaB, C.Y. (1975) A study of cardioto~n principles from the venom of

Bunganu fasciatut. Tarirnn 13, 189.

Lu,H.S. and Lo, T.B. (1974) (~romatographicseparationofBungancsfaaciantsvenom andpreliminary charao-terization of its components. l.Chin. Biodran. Soc. 3,57.

Lu, H.S. end Lo, T.B. (1978) Complete amino acid sequence of a new type ofcardiotmdn ofBwtgarusfarciatur

venom.Int.J.P~pttde Prottln Rts. 12,181.

Lu, H.S. and Lo, T.B. (1981) Complete amino acid sequence of two cardiotoßn-like analogs fromBurtgarus frtaciatus(banded krait) make venom. Toxicon 19,103.

RoaaYrs, M.F., DBasss, R.A., Mnvcax, T.C. and Darn~ns, E.A. (19'T%) ~emical modification ofthe histidine nsidue in phospholipase A2(Naja raja naja).Acase of half-site reactivity. J.biol. Chem. 252,2405. SreoNa, P.N., Goi~, J., Osaso, S.G. and ISaias, R.B. (1976) ß~Buogarotoxin, a pre-synaptic t070n with

enzymatic activity.Prod. rwAa Adad. Sci. US.A. 73,178.

VIIJOHN, C.C., Vn1s~, L. andBares, D.P. (19Th Histidine andlysineresidues andthe activityofphospholipase AZ from the vetwm ofBttis gabonida . Biochim. biophys . Acts 483, 107.

TeaLa 1 . Paosrxouresa AAcmrnOFi.oNa~aemr cNCntaro~mvs

Cardioto~n' Dose per assay Specific activity (unitlmg)t

V-2 84Etg 0.41; 0.43

V-3 30Ng 1.6; 1.7

VI 130pg 0.28; 0.30

Phospholipase AZ




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