M”/<adar /mmunn/“pl~. Vol. 18. pp 125-133 Perpamon Pre5\ ILtd 1981. Printed in Great Britain.

016lL5890/8l~02014)125 502.00/O

MOLECULAR CHARACTERIZATION OF THE COMPLEMENT ACTIVATING PROTEIN IN THE VENOM OF

THE INDIAN COBRA (NAJA N. SIAMENSIS)

GilSTA EGGERTSEN,* PETER LINDt and JOHN SJoQUIST* *Department of Medical and Physiological Chemistry, The Biomedical Centre, Box 575,

S-751 23 Uppsala, Sweden, and +Institute of Biochemistry, The Biomedical Centre, Box 576, S-751 23, LJppsala, Sweden

(Firsr received 14 March 1980; in revised form 22 August 1980)

Abstract-The cobra venom factor (CVF) from Naju n. siamensis was isolated from crude freeze-dried venom by a combination of ion-exchange chromatography and gel filtration (Bio-Rex 70, Sephadex and QAE-Sephadex). The yield of isolated product was 6-8 mg per g starting material. CVF appeared as a homogenous protein band in polyacrylamide gel electrophoresis with and without SDS present. In equilibrium sedimentation analysis the protein was homogenous with a molecular weight of 133,000. Under reducing conditions three protein bands appeared in polyacrylamide gel electrophoresis in SDS with molecular weights of 71,000,48,000 and 28,000, corresponding to a total molecular weight of 147,000. All of the bands stained with basic fuchsin, suggesting the presence ofcarbohydrate. Gel filtration in 6 M quanidine hydrochloride on Sepharose 4B of reduced and alkylated material gave three peaks, each corresponding to one of the bands visible on polyacrylamide gel electrophoresis in SDS. The amino acid composition and N- terminal amino acid sequence of each peak were determined. Using a monospecific antiserum to CVF, molecules immunologically related to CVF were detected in several other elapid venoms.

INTRODUCTION

The complement system has been recognized for nearly 100 years, when Pfeiffer and Isaeff (1894) discovered the hemolytic and bactericidal properties of serum. Some years later it was found that these effects could be destroyed by treating the serum with different snake venoms, in particular that of the Indian cobra (Nu& nuja) (Flexner and Noguchi, 1902). In 1964 Vogt and Schmidt isolated from Baja nuja venom a protein which induced anaphylatoxic activity in rat serum. A partially purified fraction from cobra venom was shown by Nelson (1966) to deplete guinea pig serum of C3 in vivo and in vitro, but it had no effect on purified C3. Miiller-Eberhard and co-workers (1966) found the active principle to be a 7s protein, which together with a 5s /?-globulin in serum (now recognized as factor B) formed a bimolecular complex capable of inactivating C3 in the presence of divalent cations.

The last 10 years of research in this field has essentially clarified the mechanism behind this reaction (see Miiller-Eberhard, 1976), and it is now well established that the cobra venom factor (CVF) and factor B form a fluid phase C3jCS

Abbreviations used: CVF, cobra venom factor; PAGE. polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.

convertase which is reported to be resistant to the complement regulating systems of the organism (Lachmann and Halbwachs, 1975). CVF has been a valuable tool in the study of the complement system. However, despite its extensive use as such, very little work has been carried out on the structure of CVF. Miiller- Eberhard and Fjellstriim (1971) estimated the molecular weight at 144,000 and the carbohydrate content at 11.2%. Alper and Balavitch (1976) showed that CVF has antigenic determinants in common with human C3. They also found a molecule immunologically related to CVF in the serum of Nuju nuju, suggesting that the venom factor is an altered form of cobra C3.

The present report describes the preparation and biochemical characterization of CVF from Nuju n. siumensis venom.

MATERIALS AND METHODS

Crude, freeze-dried venoms of Nuju n. siumensis, Nuju huje, Nuju nigricollis, Ophiophugus hunnuh, Bungurus multicinctus and Bungurus coeruleus were obtained from Miami Serpentarium Laboratories, Miami, Florida, USA. Venom from Nuja niveu was supplied by Dr. Thomas Madsen.

Sephadex G-25, G-75, G-150, Sepharose 4B, QAE-Sephadex A-25 and Blue Dextran were

125

126 COSTA EGGERTSEN, PETER LIND and JOHN SJiiQUlST

purchased from Pharmacia Fine Chemicals, L’ppsala, Sweden. Bio-Rex 70 was obtained from Bio-Rad Laboratories, Richmond, CA, U.S.A.

Guanidinc hydrochloride was obtained from Fluka AG, Switzerland. Neuraminidase (Vibrio

Cll&UW. 500 LJiml) was obtained from Behringwerke AG, Marburg-Lahn, Germany.

Disc electrophoresis in polyacrylamide gels (PAGE) was performed in 7% gels using 0.05 M

Tris-glycine buffer at pH 8.3 according to Maurer (1971). Polyacrylamide gel elec-

trophoresis in sodium dodecyl sulfate (SDS- PAGE) was run in 5 and 10% gels according to Weber rt al. (1972) and in 127” gels according to Laemmli (1970). For molecular weight determinations, reduced standard proteins were used. Protein staining of gels was done with Coomassie Brilliant Blue. Gel scanning was

performed on a Gilford Model 2520 gel scanner at 570 nm. Carbohydrate staining of gels was

performed with basic fuchsin according to Neville and Glossman (1974).

Neuraminidase treatment of purified CVF was performed in 0.1 M ammonium acetate with 0.002 M CaCl, at 37’. 35 pg of CVF was incubated either with 0.63 U of enzyme for 45

min, or with 0.25 U for 24 hr. The specimens were then freeze-dried and analyzed on SDS-PAGE

under reducing conditions according to Laemmli

(1970). Ultracentrifugal determination of the

molecular weight of CVF was done by the conventional low speed sedimentation equilib-

rium method (Yphantis, 1964). Purified CVF was dialyzed against 0.01 M sodium phosphate buffer containing 0.15 M sodium chloride, pH 7.2 and was then diluted with the same buffer to

give absorbance values of 0.5, 0.4, 0.3 and 0.2. The analysis was run in a MSE Centriscan 75 Ultracentrifuge equipped with a photoelectric scanner in a six-hole rotor at 6495 rev/min.

Reduction and S-carboxymethylation of CVF was performed under Nz with a ten-fold excess of

dithioerythrol in 6 A4 guanidine hydrochloride buffered with 0.86 A4 Tris-HCl, pH 8.6, containing 0.33;, (w/v) EDTA. Carboxymethyl- ation was done with ‘“C-iodoacetic acid.

Chromatography in 6 I%I guanidine hydro- chloride wascarried out on Sepharose4B in acolumn measuring 1.6 x 90 cm according to Fish et al. (1969). For analytical chromatography the following reduced and S-carboxymethylated standard proteins, labelled with 3H-iodoacetic acid were used: porcine phosphorylase b, human serum albumin, bovine cr-chymotrypsin A and

bovine ribonuclease A. They were run either simultaneously with CVF or separately, and blue dextran was used as an indicator of the void volume. Fractions were counted in a Packard 2425 liquid scintillation counter equipped with a

Compucorp 327 Scientist printer, programmed to calculate disintegrations per min and correct for spill-over from the *“C-channel to the 3H-channel. The molecular weight was calculated according to Porath (1963). Preparative chromatography was run in the same column without calibration proteins and blue dextran.

Carbohydrate determination was performed

by gas chromatography according to Sawardeker et al. (1965). To remove contamin-

ating carbohydrate before analysis, the speci-

mens were chromatographed on hydroxyl-

apatite in 5 mA4 potassium phosphate, pH 6.9,

and eluted with 0.25 A4 potassium phosphate. Phospholipase was assayed according to

Dennis (1973). Purified lecithin from egg yolk was supplied by Dr. David Eaker, Institute of Biochemistry, LJniversity of Uppsala. A semi-

purified phospholipase A, (Karlsson & Pongsawasdi 1980) from Naja n. sianzensis was used as standard.

Amino acid analysis was performed on a Beckman 121 M amino acid analyser according

to Spackman et al. (1958). Samples containing 200-600 pg of protein were hydrolysed in 6 M

HCl containing 1% phenol w/v at 110’ for 24 or

72 hr. Automatic Edman degradation was done with

a Beckman 890C liquid phase sequencer using a program described by Thomsen et al. (1976). Amino acid phenylthiohydantoins were

identified by high pressure liquid chromato- graphy using a Waters dual 6000A pump liquid chromatography system (Waters Assoc. Milford, MA). The analysis was performed on a

p Bondapak Cl8 column (3.9 x 300 mm) in 0.03 A4 sodium acetate, pH 5.0, using a linear gradient of acetonitrile (18-40”~ v/v, 7 min) at 37“ and a

flow rate of 2 ml per min. For separation of the hydantoins of isoleucine from phenylalanine and valine from methionine, a linear gradient of

methanol (40-59”;, v/v, 15 min) was used. The column was monitored at 254 nm. Yields of amino acid phenylhydantoins were calculated by automated integration of chromatograms coupled to the Waters system.

Purified human C3 was prepared as described by Lundwall et al. (1978). Human C3d was prepared to purity according to Bokisch ct al.

Cobra Venom Factor (CVF) 127

Fig. 1. Sephadex G-150 gel filtration in 0.1 M ammonium acetate of the void peak from Sephadex G-75 (see insertion). CVF was identified in the second peak. Insert: Sephadex G- 75 gel filtration in 0.1 M ammonium acetate of the complement inactivating material from the Bio-Rex 70 chromatography (Karlsson and Pongsawasdi, 1980; reproduced with authors’ permission). AC: phosphilipases

with anti-coagulant properties.

(1969). Antisera against CVF and human C3 and C3d were raised in rabbits by intradermal injection of 0.5-1.0 mg of the purified protein emulsified in Freund’s complete adjuvant. Booster injections were given subcutaneously in Freund’s incomplete adjuvant 4-6 weeks later. The antiserum against C3 reacted in immunoelectrophoresis with native C3 and the C3c and C3d fragments, while the antiserum against C3d reacted strongly with C3d.

Immunodiffusion and immunoelectrophoresis were carried out on microscope slides in 1% agarose (Indubiose, Pharmindustrie, Clichy, France) in 10 mMTris-acetic buffer containing 1 mM EDTA, pH 8.6. Electroimmunoassay was performed according to Laurel1 (1972) in 1% agarose and 80 mM Tris-acetic buffer containing 10 mM EDTA, pH 8.6. Determina- tion of the activity of CVF was performed by adding test material to normal human serum, which was then incubated at 37” for 60 min.

A280

CVF

whereafter the residual complement activity was estimated by CH,,-titration (Mayer, 1967).

RESULTS

Purification of CVF was performed in connection with preparation of snake venom neurotoxins (Karlsson et al., 1971). Crude, freeze-dried venom from Naja naja siamensis was dissolved in 0.08 M ammonium acetate buffer, pH 6.5. The material was then applied on a Bio- Rex 70 column equilibrated with the same buffer. The complement-depleting activity was found in the break-through fraction, which was pooled and chromatographed on Sephadex G-75 in 0.1 M ammonium acetate. The complement inhibit- ing material appeared in the void peak, while the fractions AC contain phospholipases A with anticoagulant activity (Karlsson and Pongsaw- asdi, 1980) (Fig. 1). The void material was concentrated on a Diaflo PM 10 membrane and applied on a Sephadex G-l 50 column equili- brated with the same buffer. Two peaks emerged, one in the void volume, both of which showed complement inactivation (Fig. 1). Only the second peak produced a marked C3-conversion in normal human serum in contrast to the first one, which affected C3 to a minor extent as judged by immunoelectrophoresis. PAGE in 7% gels indicated that the void material was of very high molecular weight, since it did not enter the gel. This material most probably represents the high molecular weight fraction of the venom that was earlier reported to inactivate Cl (Ballow and Cochrane, 1969). The second peak was shown by PAGE to contain, except CVF, a couple of weakly stained bands with slower electro- phoretic mobility. The fractions of this peak were

aI .

m SW sml VOLUME lmll

Fig. 2. QAE-Sephadex chromatography of the CVF-containing material from Sephadex G-150. The material was applied in 0.05 MN-ethyl-morpholine buffer, pH 7.5, and was then eluted with a linear gradient of2 x 300 ml from zero to 1.5 Mammonium acetate. CVF started to elute at approximately 10 mS. Column

volume 125 ml.

128 GiiSTA EGGERTSEN, PETER LIND and JOHN SJiiQUIST

Table 1. Purification and yield of CVF. The amount of CVF was determined by electroimmunoassay (LaurelI, 1972).

Material Total CVF (mg) Yield (7”)

Crude venom (3g) 87.6 100 Sephadex G-75 pool 48.4 46 Sephadex G- 150 pool 31.7 36 QAE-Sephadex pool 16.5 19

dialysed against 0.05 A4 N-ethyl-morpholine

acetic acid buffer, pH 7.5, and chromatographed on QAE-Sephadex A-25 with a linear gradient

from zero to 1.5 M ammonium acetate (Fig. 2). CVF eluted at 10 mS conductivity. The material

was dialysed against 0.1 M ammonium acetate, concentrated on Diaflo PM 10 and stored at -20”. The yield as determined by electro- immunoassay was approx. 20”;) of the amount

present in crude venom, or 6-8 mg CVF per g of starting material (Table 1).

The purified CVF appeared as a single

w

homogenous band on PAGE in 7% gels and on 5% gels in SDS under non-reducing conditions (Fig. 3). After reduction, three distinct bands appeared with estimated molecular weights of 71,000, 48,000 and 28,000, and which were designated to c(, /J and 1’ respectively. To verify that each band represented a definite polypeptide chain the gel was scanned at 540 nm and the absorbances of the stained bands were calculated. As shown in Table 2, the ratios of the amounts of stain correlated well with the

molecular weight ratios for each of the polypeptide chains. The protein bands were also stainable with basic fuchsin, suggesting the presence of carbohydrate in all three chains. When SDS-PAGE according to Laemmli (1970) was performed under reducing conditions the

most rapidly migrating component (y) was resolved into a tight group of three bands (Fig. 3:4). The migration pattern was not

1 2 3 4 Fig. 3. Disc electrophoresis in polyacrylamide gels of purified CVF. 1. Nondenaturating conditions in 7% gel. 2. 10% gel with SDS without reduction. 3. 10% gel with SDS under reducing conditions. 4. 12% gel with

SDS according to Laemmli (1970) under reducing conditions.

Cobra Venom Factor (CVF) 129

Table 2. Comparison of molecular weight and staining ratios of component chains of CVF after SDS-PAGE

Chains Molecular weight ratio Stain ratio

a/B 71 000/48 000 = 1.47 1.39 u/r 71 OOOj28 000 = 2.53 2.50

B/v 48 000/28 000 = 1.71 1.81

affected by treatment of CVF with neuraminidase before the electrophoresis.

The carbohydrate content of the whole molecule was determined at 3.9% (specified in

Table 3), excluding sialic acid, which did not exceed 1% (data not shown). The specific absorbance (A:$) of CVF determined in 0.1 M ammonium acetate was 10.7. By immunodif- fusion one precipitation arc was produced against the crude venom with anti-CVF, showing complete identity with the arc of the purified

product. No precipitation line appeared with the high molecular weight fraction from the G-150 fraction step. The amino acid composition of purified CVF is shown in Table 4. The phospholipase content was found to be approx. 3% or less (w/w) when compared to semi-purified phospholipase A, from the venom.

The molecular weight of CVF was determined by low speed sedimentation equilibrium centrifugation. The apparent molecular weight at the four concentrations used was obtained from the slope of In C (measured as the absorbance at 280 nm) plotted versus r2 (Yphantis, 1964). The partial specific volume was 0.734, calculated from the amino acid composition (Cohn and Edsall, 1943). The molecular weight of CVF was calculated from a plot of the apparent molecular weight versus the protein concentration by extrapolation to zero concentration. A value of 133,000 was found,

which is in accordance with the value of 147,000 from SDS-PAGE.

When chromatographed on Sepharose 4B in 6 M guanidine hydrochloride purified non-

reduced CVF eluted as a single peak with a Kd value of 0.176. Reduced and alkylated CVF

Table 3. The carbohydrate content (excluding sialic acid) of intact CVF

Carbohydrate % (w/w)

Fucose 1.2 Mannose 0.9 Galactose 0.7 Glucose 0.5 N-acetyl-glucosamine 0.6 N-acetyl-galactosamine Trace

Sum: 3.9

Table 4. Amino acid composition of intact CVF and the three isolated components, expressed as residues of amino

acid per 100 residues

Amino acid Intact CVF Chain a

LYS His

Arg Cm-Cys Asx Thr* Ser* Glx Pro

GUY Ala

Cysf Valt Met Ilet Leut

Tyr Phet

6.8 6.3 7.3 7.0 2.1 1.9 2.2 1.1 4.0 4.0 4.2 5.0

0.6 3.0 1.2 11.5 11.1 12.9 9.8 6.9 8.1 6.5 5.5 6.1 6.4 5.5 7.7

11.0 9.2 12.4 12.6 5.1 6.2 3.6 5.2 5.7 6.9 5.2 5.3 5.6 5.9 4.9 5.4 1.3 8.7 9.3 6.7 10.0 2.1 2.0 2.4 1.8 6.7 5.7 7.0 8.4 8.3 7.9 8.9 7.4 4.2 4.1 4.2 3.0 3.9 4.4 3.3 3.6

*Determined after extrapolation to zero times of values from 24 and 72 hr hydrolysis.

t Determined from 72 hr analysis. $Determined as cysteic acid after performic acid

oxidation.

(14C-iodoacetic acid) gave three peaks (Fig. 4). The highest UV-absorbance was found in the

first peak, while the radioactive labelling was most prominent in the second one. The molecular weights of the CI, /I and y-chains were calculated to be 94,000, 59,000 and 35,000, respectively. The material in each peak was pooled and transferred into 1 .O M acetic acid by

chromatography on a short Sephadex G-25 column. No significant loss of protein was observed during this procedure. The material was then freeze-dried and aliquots from each

pool were dissolved in 0.01 A4 phosphate buffer containing 1% SDS and 7 M urea, pH 7.0, and

were analysed by PAGE in 5 and 10% gels in SDS. The material of each peak migrated as a single protein band in the gels, with mobilities

corresponding to the analogous bands obtained by PAGE of whole CVF under reducing conditions in SDS.

The amino acid compositions of the individual chains are given in Table 4. As can be seen, most of the cysteine residues are in the P-chain, in good agreement with the pattern of the radioactive label. The most frequently occurring amino acids are glutamic acid and aspartic acid, except in the

a-chain, where valine slightly exceeds glutamic acid.

The amino-terminal sequences of the different

chains were determined. 20-25 nmoles of each chain was subjected to automatic Edman

130 GijSTA EGGERTSEN. PETER LIND and JOHN SJcjQL’IS’I

A280 dpm T “ID aL_a “I’ a30 3000

1

4

A

VOLUME (ml)

Fig. 4. Chromatography on Sepharosc 4B of reduced and alkylated CVF in 6 M ruanidinc hvdrochloride. The void volume was marked by Blue Dextran. and the total volume was taken as the elution volume of unbound ‘“C-labelled

iodoacetic acid.

degradation. The sequences are shown in Fig. 5. To investigate whether other elapid venoms

contain mokcules related to CVF of Nuju II.

siamensis, other snake venoms dissolved in 0.15 M NaCl at concentrations of 50 mg per ml were

tested by immunodiffusion against anti-CVF. A single precipitation arc was produced against

venom from Nuju huje, Nuju niveu, Nuju

nigricollis and Ophiophugus hunnuh. Venoms from Bungarus coeruleus and Bungurus

mufticinctus did not react. The precipitates showed complete identity with purified CVF

(Fig. 6). The antiserum against CVF failed to give precipitates with purified human C3 and normal human serum. Neither did any reaction appear between CVF and anti-human C3 or anti-

human C3d.

DISCUSSION

The results presented here suggest that CVF of

Nuju n. siamensis is a glycoprotein consisting of three polypeptide chains linked together by disulfide bonds. The amino acid composition is similar to that reported by Miller-Eberhard and

1 5 IO c( Ala-Leu-Tyr-Thr-Leu-Ile-Thr-Pro-Ala-Val-Leu-Arg-

15 20 25 Thr-Asp-Thr-Glu-Glu-Glu-Ile-Leu-Val-Glu-Ala-His-Gly

I 5 IO

B Glu-Ile-GlN-Met-Pro-Thr-His-Lys-Asp-Leu-AsN- I5 20 23

Leu-Asp-Ile-Thr-Glu-Glu-Leu-Pro-Asp-Arg-Glu-Val

1 5 IO

1; Asp-Arg-AsN-Glu-Asp-Cily-llc~Phe-Ilc,’Phe-Ala-Asp- Ser-Asp

Fig. 5. Amino-terminal sequences of the three polypeptide chains of CVF. Ile/Phe indicates that either isoleucine or

phenylalanine OCCUTS in the actual position

Fjellstriim (1971). The carbohydrate content was given by them as I 1.2”,,, whereas we have found a value of approx. S”,,. The difference can be explained by the pretreatment of our specimen on a hydroxylapatite column, a procedure which removes polysaccharides originating from the Sephadex gels during purification. The molecular weight value presented by Miiller- Eberhard and Fjellstriim is similar to our values

from the low speed sedimentation equilibrium analysis and SDS-PAGE. The results from the chromatography in 6 M guanidine hydro- chloride. however, show much higher figures, giving a value of 1 XX.000 for the intact molecule.

The reason for this is obscure. It has been reported that carbohydrate-containing polypep- tide chains may product aberrant values on gel chromatography in 6 M guanidine hydro- chloride (Fohlman er cl/. 1976) depending on the location and structure of the carbohydrate moieties present. The results of the SDS-PAGE and ultracentrifugation indicate that the molecular weight of CVF is in the vicinity of 140,000.

Recently, Pepys ct ul. (1979) suggested that CVF from N~ju n~@l consists of two polypeptide

chains. They reported that in SDS-PAGE the smallest component of CVF appeared as three bands with molecular weights ranging from 29.000 to 31,000. in agreement with our result. They interpreted the microheterogeneity as

representing protein cleavage products, which together with the /I-chain should form a counterpart of the x-chain of human C3. However, the N-terminal sequence of the y-chain was found by us to be homogcnous. Micro-

heterogeneity might reflect differences in associated carbohydrate moieties. It might also be due to small differences in the carboxy- terminal sequence. which we have not investigated. Actually. a microheterogeneity of the intact molecule has been described by Johnson and Kucich (1977). who related this to differences in the content of terminal sialic acid. As reported here. the pattern in SDS-PAGE is not affected by pretreatment of CVF with neuraminidase.

The analogy between CVF and human C3 was based on the cross-reaction between anti-CVF and human C3 (Alper- and Balavitch, 1976). We have not been able to contirm this result. One explanation for the discrepancy could be the properties of the antisera used, since individual animals differ in their antibody-producing ability with regard to both quantity and

Cobra Venom Factor (CVF)

Fig. 6. Immunodiffusion analysis. Upper: Analysis of different elapid venoms against anti-CVF. 1. CVF, 1 mg/ml. 2. Bungarusmulticinctus. 3. Bungarus coeruleus. 4. Naja nigricollis. 5. Naja nivea. 6. Naja haje. Ascan be seen, all the tested cobra venom were positive (Ophiophagus hannah not shown), while the venoms of the kraits failed to given any precipitates. Lower: Analysis of human C3 and normal human serum against anti- CVF. 1. CVF 1 mg/ml. 2. High molecular weight fraction from Naja naja venom. 3. Normal human serum withconverted C3.4. Purified human C3.5. Normal human serum. Only with CVF is a precipitate produced.

specificity. A positive precipitation reaction was also found in immunodiffusion with serum from Naja nuju and anti-CVF (data not shown) in accordance with Alper and Balavitch (1976).

Functionally, human C3b and CVF have at least two properties in common; binding to factor B and binding of CS. They differ with

regard to sensitivity to complement regulating proteins (Lachmann and Halbwachs, 1975) and

binding to cell receptors (Pepys, 1976). The structural data for CVF reported here reveal some differences between it and C3 which are also reflected in the difficulties of producing

cross-reacting antibodies. The three-chain structure of CVF proposed by us could either indicate that one chain of C3 has been eliminated during evolution, or that CVF is produced with two chains, one of which is

132 COSTA EGGERTSEN, PETER LIND and JOHN SJijQUIS’l

cleaved during the process of secretion. Proteolytic cleavage of CVF in the venom is less probable, since elapid venoms contain very little, if any, proteolytic activity (Mebs, 1970; Geiger and Kortman, 1977).

The N-terminal sequences of the chains of C3

and C3b have recently been published by Tack et al. (1979). Comparison with the N-terminal sequences of the three CVF chains reveals no homology but considerably more sequencing must be done before homology can be ruled out

or demonstrated.

Several other elapid venoms were found to contain an immunological counterpart to CVF. This has earlier been shown for Naja haje (Shin et

al., 1969), and some characteristics of the latter factor have been described (Bauman, 1977; Vogt et a/., 1979). The inactivation of complement factors in normal human serum by crude venoms in vitro also suggests the presence of CVF-like

activities in elapid venoms (Eggertsen et al.,

1980).

Acknowledgements-We are in great debt to Dr. J. Lonngren at the Dept. of Organic Chemistry, Arrhenius Laboratory, University of Stockholm, for performing the carbohydrate analysis, and to Mr. Ake Engstriim for performing the automatic Edman degradation. We are also indebted to Dr. Evert Karlsson for help in preparing CVF and to Dr. David Eaker and ass. Ake Lundwail for valuable discussions. This investigation was supported by grant from Swedish Medical Research Council (Project number 13X-2518).

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