Journal of Immunological Methods, 30 (1979) 105--117 © Elsevier/North-Holland Biomedical Press

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AN IMPROVED METHOD F O R THE I S O L A T I O N FROM N A J A N A J A VENOM OF COBRA F A C T O R (CoF) F R E E OF PHOSPHOLIPASE A

M.B. PEPYS, C H R I S T I N E T O M P K I N S

1 and A.D. S M I T H 2

Immunological Medicine Unit, Department of Medicine, Royal Postgraduate Medical School, 1 Department of Neurology, Royal Free Hospital School of Medicine, and 2 Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London, U.K. (Received 12 March 1979, accepted 16 May 1979)

An improved method is reported for the isolation from cobra (Naja naja) venom of cobra factor (CoF), the antieomplementary protein which is derived from cobra C3. Sequential chromatography on DEAE-Sepharose, Sephacryl-S200, and finally hydroxylapatite yielded 6.25 mg CoF per gram of crude venom. The purified CoF had 1 unit of functional anticomplementary activity per 1--2/~g of protein, and was homogeneous on gradient and non-reduced sodium dodeeyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE). In SDS-PAGE after reduction with mercaptoethanol there were two major bands (M.W. 75,000 and 51,000 daltons), three minor bands (M.W. 29--31,500 daltons) and two trace bands (36,500 and 41,500 daltons). By analogy with mammalian C3 it is suggested that the CoF consists of two polypeptide chains linked by disulphide bridges, one of which undergoes cleavage of the peptide chain at several points either in vivo or in vitro.

INTRODUCTION

The major a n t i c o m p l e m e n t a r y protein o f cobra (Na]a naja) venom, know n as ~cobra factor (CoF) (Ballow and Cochrane, 1969; Miiller-Eberhard and FjellstrSm, 1971) is a glycoprotein which is widely used to inactivate comp l e m en t in vitro. It is also the most effective experimental agent for inducing in vivo c o m p l e m e n t depletion and as such has been used to demonstrate the role Of c o m p l e m e n t in various i n f l a m m a t o r y and immunological reactions (Cochrane et al., 1970; Hill and Ward, 1971; Fong and G ood, 1971), and in induction o f t h y m u s - d e p e n d e n t a n t i b o d y f o r m a t i o n (Pepys, 1972, 1974, 1976). Before attributing in vivo or in vitro effects o f CoF preparations to their action on c o m p l e m e n t , it is necessary to exclude participation of ot her biologically active molecules which m a y be present as contaminants. The presence has been r e p o r t e d o f three distinct 'activities' in CoF preparations which are n o t related to the interaction of the CoF molecule itself with the c o m p l e m e n t system. These activities are: phospholipase A activity (Lachmann et al., 1976}, a l y m p h o c y t o t o x i c activity attributable to a low molecular weight (13 K daltons) protein (Ballow et al., 1973) and an adjuvant

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activity which interferes with induction of specific immunological tolerance in vivo (Morrison et al., 1976). We have previously reported isolation from Naja naja venom of CoF which was completely free of phospholipase A, l y m p h o c y t o t o x i c and adjuvant activities (Pepys, 1974, 1976; Pepys and Taussig, 1974). The m e t h o d used was sequential chromatography of the venom on DEAE cellulose and Sephadex G-200, according to Ballow and Cochrane (1969) and the details are presented here. However, fractionation of later batches of cobra venom by the identical procedure sometimes failed to remove all phospholipase A activity from the CoF. As Lachmann et al. (1976) have demonstrated, this is due to the presence of phospholipase A activity associated with a molecular species of a size comparable with CoF. We now report an improved procedure for isolation of CoF using ionexchange, gel filtration and h y d o x y l a p a t i t e chromatography. The m e t h o d is simple, requires only column chromatography apparatus and yields a product devoid of phospholipase A. The polypeptide chain composition of the purified CoF has been characterised. M A T E R I A L S AND METHODS

Cobra venom Lots of venom, product number V-9125, were obtained from Sigma L o n d o n Chemical Co. Ltd., Surrey, England as follows: 1974, Na]a naja venom lots 73C-1380 and 123C-0670; 1975, lot 124C-0339; 1977 and 1978, Na]a naja kaouthia venom, lot 15C-0223.

Assay procedures Anticomplementary activity of CoF was measured according to Ballow and Cochrane (1969). Samples (0.1 ml) of material to be tested were incubated for 20 min at 37°C with 0.4 ml of a 1/20 dilution of fresh normal human sera in complement fixation test diluent (CFT) (Oxoid Ltd., London). A 0.4 ml aliquot of a 2% suspension in CFT of sheep erythrocytes (E) sensitised with 6 minimal haemolytic doses of IgM rabbit anti-E antibody (Lachmann and Hobart, 1978) was then added and the mixture incubated for a further 20 min at 37°C. After the addition of 2 ml of cold phosphate buffered isotonic saline, pH 7.2 (PBS, Oxoid Ltd., London), the tubes were centrifuged (500 X g, 10 min) at 4°C, and the optical density of the supernatant measured at 541 nm. The percentage haemolysis was calculated from this value. The unit of CoF activity was defined according to Ballow and Cochrane (1969) as the quantity of CoF in 0.1 ml which caused 50% inhibition of lysis in the above assay. Isolated preparations of CoF were titrated in 0.1 ml volumes in CFT to determine their activity. The concentration of CoF as antigen was measured by electroimmunoassay (Laurell, 1972) using a monospecific rabbit anti-CoF serum in the gel. The results expressed as 'antigenic units' represent the peak heights in mm of the rockets.

107 The protein content of isolated CoF preparations was estimated from their optical density at 280 nm assuming ~lcm~l~= 10.0, based on the value of 9.7 for human C3 (Tack and Prahl, 1976). Phospholipase A (PLA) activity was sought and quantitated b y a modification of Hanahan et al.'s method (1954). 50-gg samples in Tris/HCI buffer, pH 6.5, were incubated at 25°C with 2 mg ovolecithin (Lipid Products, South Nutfield, Surrey) in 2 ml ether together with 10 t~l of 250 mM Ca 2÷. After periods varying between 1 h, for rough testing, and ~ days, for exhaustive confirmation of the absence of PLA, the mixtures were submitted to thin layer chromatography and assay of the phosphorus in any lysolecithin produced. PLA activity was calculated as nM lysolecithin generated per h. The concentration of C3 in mouse serum samples was measured by electroimmunoassay with a monospecific sheep anti-mouse C3 serum as previously described (Pepys, 1975). The assay was calibrated using isolated mouse C3 (Pepys et al., 1977).

Isolation procedures ( l a) DEAE-cellulose chromatography 4.1 g of lot 123C-0670 and 4.0 g of lot 73C-1380 were pooled, dissolved in 400 ml of 0.01 M sodium phosphate buffer, pH 7.5, and p u m p e d onto a 90 X 5.0 cm column of DEAE cellulose (DE52, Whatman) equilibrated with the same buffer. The column was then eluted at 200 ml/h with the starting buffer until the A280 of the effluent returned to zero. A linear 10 1 : 10 1 gradient of starting buffer and limit buffer (0.01 M phosphate, pH 7.5, containing 0.5 M NaC1) was then p u m p e d on at 160 ml/h, and 20 ml fractions were collected. Fractions were tested for anti-complementary activity and CoF antigen, and all those which produced up to 50% inhibition of heamolysis were pooled and concentrated to 25 ml on an Amicon PM30 membrane in a stirred Diaflo cell (Amicon Ltd., Bucks).

(1 b) Sephadex G-200 gel filtration The whole CoF pool was p u m p e d onto a 90 X 5 cm column of Sephadex G-200 (fine) (Pharmacia G.B. Ltd., London) equilibrated with PBS, and eluted with PBS at 60 ml/h. Ten ml fractions were collected and ~ s a y e d fo~ anti-complementary activity and CoF antigen. The CoF was pooled conservatively, especially on the descending limb of the peak, concentrated on a PM30 membrane and stored frozen at --20 ° C.

( 2a ) D EAE-Sepharose-CL-6B chromatography Three separate batches of 4.0 g of Naja na]a kaouthia venom (lot 15C0223) were processed in the same way. Each was dissolved in 0.01 M sodium phosphate buffer, pH 7.5, containing 0.01% sodium azide, loaded onto a 24 × m column of D E A E ~ e p h a r o s e CL-6B (Pharmacia G.B. Ltd., London), eq ~ted w!th the same buffer and eluted at 200 ml/h with starting

108 buffer until the A280 of the effluent returned to normal. A linear gradient of 4 1 starting buffer : 4 l limit buffer (starting buffer containing 0.5 M NaC1) was then p u m p e d on at 160 ml/h and 13 ml fractions collected. The fractions were tested for anticomplementary activity and CoF antigen, pooled conservatively and concentrated on a PM30 membrane.

(2b) Gel filtration Portions of the CoF pool from DEAE-Sepharose were gel filtered on either Ultrogel AcA44 (LKB Instruments Ltd., London) or Sephacryl-S200 (Pharmacia G.B. Ltd., London). The volume of applied sample was less than 2% of the column volume in each case. The AcA44 column (2.6 × 90 cm) was eluted at 16 ml/h and 3.0 ml fractions were collected. The S-200 column (1.6 × 88 cm) was eluted at 5.6 ml/h and 3.0 ml fractions were collected. The fractions were tested for CoF b y antigenic assay only, pooled and concentrated on a PM30 membrane. (2c) Hydroxylapatite chromatography A 7 ml column of hydroxylapatite (Bio-Gel HT, Bio-Rad Labs., Richmond, California, U.S.A.) was poured in a 10 ml disposable plastic syringe and equilibrated with 0.01 M potassium phosphate buffer, pH 7.4, containing 0.01% sodium azide. The CoF pool from gel filtration was dialysed into the same starting buffer, p u m p e d onto the column and eluted at 48 ml/h, with collection of 3 ml fractions. When the A280 of the effluent returned to zero the column was eluted with a 90 ml : 90 ml linear gradient of starting buffer and 0.4 M potassium phosphate limit buffer, pH 7.4. The fractions were tested for CoF antigen and in gradient PAGE, and those containing only a single band representing CoF were pooled, concentrated on a PM30 membrane, dialysed into PBS and stored frozen at --20°C. Polyacrylamide gel electrophoresis Pharmacia 4--30% gradient polyacrylamide gels were run in the Pharmacia gel electrophoresis apparatus in 0.09 M Tris/0.08 M borate/0.003 M Na2 EDTA buffer, pH 8.35. Reduced and unreduced samples were analysed b y sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) asing the m e t h o d of Laemmli (1970) in 10% and 12% slab gels. Proteins used for comparison and as molecular weight markers were as follows: isolated mouse C3i (obtained from aged normal mouse serum by the m e t h o d of Pepys et al., 1977); isolated human C3 (obtained b y the m e t h o d of Tack and Prahl, 1976), a-chain (120 K), ~-chain (75 K daltons) (Bokisch et al., 1975); phosphorylase a (94 K daltons), pyruvate kinase (57 K daltons) and ovalbumin (43 K daltons) from Sigma Ltd.; human IgG (heavy chain, 51 K; light chain, 26 K daltons). Isolated human C3 was digested with trypsin to form C3b and C3c + C3d as described by Bokisch et al. (1969). Effect of CoF on mouse C3 in vivo Individual adult CBA/Ca male mice from OLAC Ltd., Bicester, Oxon,

109

England, and C57B1/6 female mice from Bantim and Kingman Ltd., Hull, England, all weighing a b o u t 20 g, were given intraperitoneal injections of different doses of PLA-free CoF isolated by the improved method. Some mice received a single dose and others 4 equal doses over 24 h. All animals were bled from the tail before injection of CoF, 3 or 4, 6 or 7 and 11 or 12 days later. After clotting at r o o m temperature for 4 h, the sera were separated individually and stored at --20 ° C for assay simultaneously at the end of the experiment. RESULTS

Isolation of CoF by the method of Ballow and Cochrane (1969) We initially used the method of Ballow and Cochrane (1969) involving sequential chromatography on DEAE-cellulose (Fig. 1) and Sephadex G200 (Fig. 2) to obtain isolated CoF. This material was homogeneous in PAGE at an applied sample concentration of 250 gg/ml, induced formation in rabbits and mice of a monospecific antibody and had a specific activity of 1 U/4.0-5.0 #g protein (Pepys, 1974). It was also free of PLA activity even in the exhaustive 3 day test. The low molecular weight, third peak off G200 (Fig. 2) was always rich in PLA as well as containing at least three other components detected in electroimmunoassay with rabbit antiserum raised

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Fig. 2. Elution profile of CoF pool from DEAE cellulose on 8ephadex G200. against c r u d e C o F o f f D E A E cellulose. H o w e v e r in 1 9 7 5 , using S i g m a V - 9 1 2 5 l o t 1 2 4 C - 0 3 3 9 o f Naja naja v e n o m , t h e C o F o b t a i n e d b y t h e s a m e p r o c e d u r e was c o n t a m i n a t e d w i t h s o m e P L A . R e p e a t e d gel f i l t r a t i o n o n Ultrogel A c A 4 4 , w h i c h y i e l d e d a single a p p a r e n t l y s y m m e t r i c a l p r o t e i n p e a k c o n s i s t i n g o f C o F , did n o t r e m o v e t h e P L A a c t i v i t y , suggesting t h e e x i s t e n c e o f a species o f P L A w i t h e i t h e r a c a p a c i t y t o associate w i t h C o F or a c o m p a r a b l e m o l e c u l a r size ( L a c h m a n n et al., 1 9 7 6 ) .

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Isolation o f CoF by the improved method Fractionation of Na]a naja kaouthia venom on DEAE-Sepharose-CL-6B (Fig. 3) produced greater resolution of CoF from other proteins than was seen with DEAE-cellulose, although since different lots of venom were used it is n o t possible to attribute the different profiles only to the chromatography medium. The separated CoF was gel filtered on either Ultrogel AcA44 (Fig. 4) or Sephacryl S-200 (Fig. 5), b u t the fractions containing CoF still had some PLA activity. The CoF was therefore subjected to a further purification step on hydroxylapatite. Under the starting conditions a broad flat peak was eluted (Fig. 6) which contained PLA activity. Application of a linear phosphate gradient then eluted the CoF in apparently pure form (Figs. 7 and 8). CoF derived from both AcA44 and $200 columns gave exactly the same elution profile on hydroxylapatite, b u t after concentration and exhaustive (3 day) testing for PLA the material which had been filtered on $200 was free of PLA whilst that from AcA44 showed trace residual contamination. Crude venom at 1 pg/ml contained PLA activity of 69 nmoles/h. The yield of pure CoF after DEAE-Sepharose, S-200 and hydroxylapatite columns was 6.25 mg/g of venom. Analysis in gradient PAGE of the concentrated CoF pools after each of the three column procedures is shown in Fig. 7. The 4--30% gels used here only retain molecules larger than 50,000 daltons, so no low molecular weight components are shown by this technique. The major band in each case represents the CoF, whilst the main contaminant in the CoF pool after gel filtration was present in the material which did n o t bind to hydroxylapatite. Characterisation o f CoF isolated by the improved method The isolated CoF off hydroxylapatite produced a single band in gradient PAGE (Fig. 7c) and in non-reduced SDS-PAGE (Fig. 8). After reduction

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Fig. 8. Non-reduced 12% SDS-PAGE analysis o f three batches o f C o F isolated on D E A E Sepharose, Sephacryl S-200 and hydroxylapatite. Fig. 9. R e d u c e d 10% SDS-PAGE analysis of isolated CoF. a, m o u s e C3i; b, C o F showing the t w o m a j o r bands and the group of three m i n o r bands; c, h u m a n C3c + C3d; d, h u m a n C3b; e, native h u m a n C3.

with mercaptoethanol, CoF from different lots of venom was run in SDSPAGE and yielded bands with the following apparent molecular weights: t w o major bands, 75 K and 51 K daltons respectively, a group of three minor bands between 29 and 31.5 K daltons, and t w o trace bands, 36.5 K and 41.5 K daltons respectively (Figs. 9 and 10). CoF shows immunochemical cross-reactivity with human C3 (Alper and Balavitch, 1976) and interacts in the mammalian complement sequence like autologous C3b which is insus-

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ceptible to C3bINA ( L achm a nn and Nicol, 1973). We t h e r e f o r e com pared the p o l y p e p t i d e chain structure with t h a t of h u m a n C3, C3b, C3c + C3d and mouse C3i (Fig. 9). CoF, mouse C3i and h u m a n C3 digested with trypsin to yield C3c and C3d resembled each other, there being in each case two main bands o f larger molecular weight and a series of minor bands of smaller apparent size. E f f ect o f PLA-free CoF on mouse C3 levels in vivo The effect on serum C3 of intraperitoneal injection of CoF in t w o strains of mice is shown in Fig. 11. The CoF caused no noticeable ill effects even in

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Days Fig. 11. Effect of CoF, isolated by the improved method, on mouse C3 in vivo. The serum C3 levels are shown of individual mice treated as follows. One dose of CoF i.p. on day 0, CBA/Ca mice: o, 1 U; o, 2 U; D, 4 U; n, 8 U ; 0 , 25 U ; 0 , 50 U;A. 100 U. C57B1/6 mice: A, 1 U; v, 2 U; v, 4 U; (}, 8 U; ~, 50 U;