The direct lytic factor of cobra venom: purification and chemical characterization

BIOCHIMICA ET BIOPHYSICA ACTA

53

BBA 3 5 1 5 3

T H E D I R E C T LYTIC FACTOR OF COBRA VENOM; P U R I F I C A T I O N AND CHEMICAL C H A R A C T E R I Z A T I O N

S A R A H A L O O F - H I R S C H , A N D R E DE V R I E S AND A R I E H B E R G E R

The Rogoff-Wellcome Medical Research Institute, Tel Aviv University and Beilinson Medical Center, Petah-Tikva (Israel), and Department of Biophysics, The Weizmann Institute of Science, Rehovoth (Israel) (Received A u g u s t i8th, 1967)

SUMMARY

The direct lytic factor (DLF) from the venom of Ringhals (Haemachatus haemachates) was isolated and purified b y successive application of trichloroacetic acid and salt precipitation. The purified D L F is homogeneous b y ultracentrifugal and electrophoretic criteria and is about twice as active as the material isolated previously by paper electrophoresis. D L F is a single-chain protein with a molecular weight of 7000. I t contains 57 amino acid residues cross-linked intramolecularly by 4 disulfide bridges. Leucine is in the amino terminal position, and serine in the carboxyl terminal position of the protein. D L F causes inhibition of growth in some bacteria.

INTRODUCTION

Snake venoms have been classified into two major groups according to the mode of their hemolytic action : The direct lytic venoms, which are capable of hemolysing washed red blood cells, and the indirect ones, which fail to do so unless supplemented with a source of phosphatides 1. Lysis of washed erythrocytes b y cobra venoms is produced by the synergistic action of two venom components: A basic protein which is moderately hemolytic by itself and therefore named 'direct lytic factor' (DLF) and the venom phospholipase A which is non-hemolytic when applied alonee, 3. The D L F enables the venom phospholipase to split the red-cell-membrane phospholipids 3. A similar synergistic action has been demonstrated on platelets 4 and mitochondria 5. The difference in sensitivity of washed red cells from various animal species to both the lytic and phospholipid splitting actions of Ringhals venom is primarily a reflection of their susceptibility to the action of D L F 6. The binding of 131I-labeled D L F to red blood cells varies with the type of the erythrocyteL Abbreviation: DLF, direct lytic factor.

Biochim. Biophys. dcta, 154 (1968) 53-60

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S. ALOOF-HIRSCH, A. DE VRIES, A. BERGER

The present study is concerned with the direct lytic factor of the venom of Ringhals (Haemachatus haemachates). In previous studies the DLF was separated from whole venom by paper electrophoresis. Material thus obtained is, however, a mixture containing a number of components. In this report we describe the purification of the Ringhals D L F and present some of the chemical and physicochemica 1 characteristics of the pure protein. In addition, the growth-inhibiting effect of DLF on certain bacteria is reported. MATERIALS AND METHODS

Venom

Ringhals (H. haemachates) venom was obtained from Koch-Light Laboratories, England.

Electrophoresis Paper electrophoresis was performed in a conventional apparatus using 0.05 M sodium phosphate buffer (pH 7.0). A sample containing 2OO-lOOO/,g protein in lO-2O #1 was streaked along a 1.5-cm line across the middle of a strip of Whatman No. I paper. The electrophoresis was carried out for 16 h applying a voltage of about 4 V/cm. Disc electrophoresis on acrylamide gel was carried out by the method of REISFELD, LEWIS AND WILLIAMS8. The sample of 5o-2oo#g protein in o. 1-o.2 ml ofo.25 M sucrose was layered on the spacer gel after the anode compartment was filled with buffer. Electrophoresis was carried out by applying a current of 8 mA per tube for 45 min.

Protein concentration Protein concentrations were determined according to the method of LowRY

et al2. The absorbance at 660 m/~ of a mixture (final vol. 3.6 ml) containing 2o-200/*g protein was read after a time interval of 30 min in a Beckman DB spectrophotometer using cuvettes of I cm. The absorbance of IOO/,g of pure D L F preparation was 0.36.

Spectra Spectra were determined in a Beckman DB recording spectrophotometer using i-cm cuvettes.

Sedimentation and diffusion measurements Sedimentation analyses were carried out in a Spinco model E ultracentrifuge, at 2o-22 °, with the schlieren optical system. The samples were sedimented in a synthetic boundary cell 1° at 56 IOO rev./min for the determination of the sedimentation coefficient. Sedimentation was also carried out at 29 500 rev./min for molecular weight determination by the YPHANTIS midpoint technique 11. Diffusion measurements were performed in the same ultracentrifuge, in a synthetic boundary cell at 8225 rev./min.

Amino acid analyses Amino acid analyses were performed on samples of 1-2 mg protein in a Beckman Spinco automatic amino acid analyzer, model 12o B. Samples were hydrolyzed in vacuo in 6 M HC1 at I I O ° ± I ° for 22, 48 and 72 h. Separate samples were oxidized with performic acid prior to hydrolysis and used for the estimation of cystine as cysteic acid. I-I202 (o.I ml, 300/0) was added to 0. 9 ml formic acid and the resulting solution allowed to stand at room temperature in a stoppered flask; IO mg protein were dissolved in 0.2 ml of the performic acid solution thus obtained. After 2 h at room temperBiochim. Biophys. Acta, 154 (I968) 53-60

RINGHALS DIRECT LYTIC FACTOR

55

ature, 20 ml ether were added, the precipitate was collected and washed twice with 20 ml of ether to remove the performic acid. Tryptophan was analyzed colorimetrically according to SPIES AND CHAMBERS 12.

Determination of the NH~ terminal amino acid The NH~ terminal amino acid was determined both b y the dinitrophenylation and carbamylation techniques. Dinitrophenylation. Purified D L F (3 rag) was dissolved in I ml of 5 ~o NaHC03, and stirred with 2,4-dinitrofluorobenzene (0.05 ml of a IO% solution in ethanol) for 16 h in the dark at room temperature. The precipitate formed was separated by centrifugation, washed 3 times with ethanol, dissolved in I ml 6 M HC1 and hydrolyzed in vacuo at IiO ° for 22 h. The hydrolysate was diluted 6-fold with water, the dinitrophenylamino acids were extracted with ether, and the ether solution evaporated to dryness. After removal of dinitrophenol by sublimation, the DNP-amino acids were dissolved in acetone and resolved by two-dimensional thin-layer chromatography, according to RANDERATH1:3. The following developing systems were used: (i) In the first direction, toluene-pyridine-ethylene chlorohydrine-o.8 M NH4OH (IOO:3O:6O: 60, by vol.). (The upper phase was used for development, the lower for pretreatment of the layer.) (2) In the second direction either (a) benzene-pyridine acetic acid (80:20:2, by vol.) or (b) chloroform-methanol acetic acid (95:5 :I, b y vol.). Further identification of the terminal DNP-amino acid was obtained by thinlayer chromatography of the methyl ester derived from it la. The silica gel containing the yellow spot was transferred from the plate into a test tube and I-rnl aliquots of diazomethane in ether were added until evolution of gas ceased. The mixture was then centrifuged, and the supernatant fluid was evaporated to dryness. The DNP-amino acid methyl ester obtained was dissolved in acetone and subjected to thin-layer chromatography. The developing system used was toluene-pyridine-acetic acid (80 :IO :I, by vol.). Carbamylation and identification via the hydantoin according to STARK AND SMYTF95). The analysis was carried out with 5 mg of protein in i ml 8 M urea at p H 8 as described. After carbamylation the sample was diluted with I ml of acetic acid and dialyzed against 50 % acetic acid. After dialysis the concentration of the carbamylated protein was determined on an aliquot (amino acid analysis after hydrolysis). The rest was submitted to cyclization, isolation and hydrolysis of hydantoins as described, and finally analyzed on the amino acid analyzer.

Determination of the COOH-terminal-amino acid Crystalline, diisopropyl-fluorophosphate-treated carboxypeptidase A was obtained from Worthington Biochemical Corp. The enzyme solution was prepared b y diluting I volume of the suspension with IO volumes of IO% LiC1 and stirring for several h at 4 °. The concentration of the enzyme was determined from absorbance measurement at 278 m# assuming that a solution of i mg/ml gives an absorbance value of 1.94 (I cm). Digestion of D L F with carboxypeptidase A was performed as follows: D L F (o.41 raM) and 0.2 mg/ml enzyme in 0.02 M ammonium bicarbonate buffer (pH 8.0) were kept at 37 °. At various times aliquots of 0.2 ml were removed and the carboxypeptidase was inactivated by the addition of o.I ml I M HC1. After centrifugation the supernatant was evaporated, the residue dissolved in citrate buffer (pH 2.2), and analyzed on the amino acid analyzer. Biochim. Biophys. Acta, 154 (1968) 53-60

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S. ALOOF-HIRSCH, A. DE VRIES, A. BERGER

Assay of hemolytic activity The hemolytic activity of the D L F was assayed by determining the amount of hemoglobin released from the erythrocytes into the suspending medium. To o.I ml of washed and packed human erythrocytes in siliconized test tubes were added various concentrations of D L F in o.I ml saline solution. The mixtures were incubated for 2 h at 37 °, diluted with cold saline to 1.5 ml, centrifuged and the supernatant measured for hemoglobin in a spectrophotometer at 54 ° m/~.

Assay of phospholipid splitting The property of D L F to enable phospholipase A to split the phospholipids in red-cell membranes was checked by paper-chromatographic analysis. To I ml of osmotic hemolysate which was prepared by adding 3 volumes of distilled water to i volume of washed and packed erythrocytes were added 300 #g D L F and 400 #g partially purified Vipera palestinae phospholipase A (CH. KLIBANSKY, private communication). The mixture was incubated for 2 h at 37 °. At the end of the incubation the ghosts were centrifuged and washed once with saline. Lipids were extracted according to the procedure of MARINETTI et al. 1G, and chromatography was carried out on silica-impregnated paper according to the method of REED et al. iv. Chromatograms were stained with Rhodamine 6G.

Effect of D L F on growth of bacteria Growth experiments were performed with Escherichia coli and Staphylococcus aureus. E. coli was grown in basal medium of DAVIS AND MINGoLIlS; S. aureus was grown in a semisynthetic medium containing casein hydrolysatO 9. Fresh overnight cultures containing o.I o/o glucose were transferred into tubes containing the appropriate medium, o.2% glucose and D L F at various concentrations. The cultures were incubated at 37 ° with shaking and their absorbance was measured at different times in a Klett colorimeter using a green filter. At 6 and 24 h a loopfull of each culture was streaked on a plate containing tryptic digest agar. RESULTS

Purification of D L F from Ringhals venom A i % solution of Ringhals venom was fractionated by differential precipitation with trichloroacetic acid. On adding concentrated aqueous trichloroacetic acid (IOO g in ioo ml solution) precipitation began at 2.5-3% (w/v). After 15 rain at room temperature the suspension was centrifuged, the precipitate was collected and the supernarant again treated in the same manner. Trichloroacetic acid concentrations were raised successively to 4, 5, 6 and 8 %. The composition of the different fractions was examined electrophoretically on paper and acrylamide gel. The precipitate obtained at 8 % trichloroacetic acid contained mainly DLF, but also significant amounts of neighboring fractions. "[he precipitate was redissolved in water and the solution repeatedly extracted with ether until no free acid could be detected in the ether extract. The trichloroacetic acid-free solution was brought to the original volume and solid (NH4)2SO4 was added to a final concentration of o.3I g per ml original solution. After 15 min at room temperature, the precipitate was collected and solid (NHa)2SO 4 was added to the supernatant to 0.35 g per ml of original solution. The two precipitates were combined, redissolved in a small amount of water and passed through a G-5o Sephadex column with o.I M acetic acid as eluent to remove (NH4) 2SO4. Biochim. Biophys. Acta, 154 (1968) 53 60

RINGHALS DIRECT LYTIC FACTOR

57

Fig. i. E l e c t r o p h o r e t i c p a t t e r n s o b t a i n e d at v a r i o u s stages of purification of D L F b y disc electrophoresis on a c r y l a m i d e gel. a, whole v e n o m ; b, trichloroacetic acid precipitates o - 3 % trichloroacetic acid; c, 3-4 ,% trichloroacetic acid; d, 5 - 6 % trichloroacetic acid; e, 6 - 8 % trichloroacetic acid; f, D L F after (NH4)2SO 4 precipitation. Fig. 2. Electrophoretic p a t t e r n s on p a p e r : a, whole v e n o m ; b, D L F .

The protein peak was isolated and lyophilized. This fraction was homogeneous when examined electrophoretically on acrylamide gel and paper (Figs. I, 2). From IOO mg whole venom 8- 9 mg purified D L F were obtained.

Spectrum Purified D L F (acetate) showed an absorption m a x i m u m at 278 m/~ with a molar extinction e ~ 2900 (absorbance of 0.42 at I mg/ml).

HemolyEc activity Purified D L F showed about twice the hemolytic activity as that of D L F isolated b y paper electrophoresis (Fig. 3).

Molecular weight Sedimentation and diffusion coefficients were measured on 0. 7 and 0.35 % solution of D L F in 0.05 veronal buffer (pH 8.o)-o.15 M NaC1. D L F sedimented as a single symmetric boundary in the ultracentrifuge. From the sedimentation coefficient,

0.6

04

o--

~b

1do Protein (pg) Fig. 3- H e m o l y t i c a c t i v i t y of crude D L F , isolated b y paper electrophoresis (&) and of purified D L F (O). E v e r y p o i n t r e p r e s e n t s t h e a v e r a g e of 3 e x p e r i m e n t s , each of w h i c h was done in duplicate. Biochim. Biophys. de:a, 154 (1968) 53-6~

5~

8. AI.OOF-HIRSCH, A. I)E VRIES, A. BERGER

S~o,w = 0.97 S, the diffusion coefficient, Dz0,u~ 13.5" IO-7 cmZ/sec and an assumed partial specific volume of o.75, a molecular weight of 7ooo was calculated. Sedimentation equilibrium runs with o. 7, o.46, o.35 and o.23% solutions of D L F in o.o5 veronal buffer (pH 8.o)-o.15 M NaC1 evaluated by the YPHANTIS midpoint method, yielded a molecular weight of 69oo. Amino acid composition The results obtained with the pure DLF fraction are given in Table I. When TABLE 1 AMINO ACID COMPOSITION OF R I N G H A L S - D L F

R e s i d u e s per 6 leueine residues.

A m i n o acid

Time of hydrolysis (h) -

Lysine Histidine Arginine A s p a r t i c acid Threonine Serine Glutamicacid Proline Glycine Alanine HMfcystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tr yptophan***

22

48

72

22*

IO,I 0.86 0.96 5.5 2.89 2.56 1.o6 4-36 1.91 I.OO 6.74 3-32 2.34 1.8 6.00 0-9 i.oo o.oo

9.6 i.oo 0.78 5 .6 2.85 2.23 i.oo 4.45 1.9o 1.o 3 6.55 4 .oo 2.2 1.94 6.00 0.87 i.oo o.oo

IO

lO.3 9-9 0.96 o.81 1.o6 5 '81 5 .82 3.41 3.27 2.73 2-56 1.5o 1.55 5.05 5.00 2.24 2.26 1.o6 1.o9 7.81" 7.88* 3 .62 4 -o8

9,2 0,76 i,o 3 5,74 3,I2 2,3 1,5o 402 2,28 1,13 7,62* 3,99

1.93 1.96 6.00 6.o0

1,93 6,00

1.o 9 0.80 o.oo o.oo

i,o o,oo

0.94 5.57 2.8 2.1 1.15 4.7 1.82 0.88 5.7 ° 3.73 2.38 1.84 6.00 0.88 0.93 o.oo

48*

Total Ammonia

Nearest integer

72? io i i 6 3 3 i 5 2 i 8 4 2 2 6 i ** i o 57

5.5

5.5

6.9

7 .o

7.3

* C y s t i n e d e t e r m i n e d as cysteic acid. ** F o u n d I . I b y difference s p e c t r u m (pH 6 vs. p H 13) of t h e n a t i v e m o l e c u l e a t 296 mp, a s s u m i n g tool. wt. 7ooo. *** T r y p t o p h a n d e t e r m i n e d c o l o r i m e t r i c a l l y . * S a m p l e o x i d i z e d w i t h p e r f o r m i e acid (see METHODS).

necessary

corrections were made

for destruction

during hydrolysis,

and when values

were rounded off to the nearest integer (last column), the amino acid composition corresponded to a molecular weight of 7076 (calculated as the acetate). Titration with p-ehloromercuribenzoate according to BOYER24 showed no free sulthydryl groups. Terminal amino acids of DL F D L F has only one amino terminal amino acid, leucine, as detected by dinitrophenylation. The yields of the amino terminal amino acid, leucine, was 71°/o recovery, based on the amounts of carbamyl compounds taken for cyclization. Carboxypeptidase A caused the release of serine only. The yields were: 0.28, 0.33, 0.42, 0.43, 0.58 moles of serine after 0.5, 2, 4, 6 and 9 h of incubation, respectively. Biochim. Biophys. Acta, 154 (1968) 53-60

RINGHALS DIRECT LYTIC FACTOR

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Splitting of red cell phospholipids Chromatographic analysis of phospholipids extracted from ghosts incubated with the pure D L F and Vipera phospholipase A showed full splitting of phosphatidyl ethanolamine, phosphatidyl serine and lecithin, with the appearance of the corresponding lyso products. Bacterial growth inhibition In the presence of 20/~g/ml D L F the cultures of both E. coli and S. aureus grew at the same rate as an untreated control culture. The growth ofS. aureus was inhibited by D L F in concentrations of 50/~g/ml and higher. A typical growth curve at IOO #g/ml D L F is given in Fig. 4I

I

3 --',

I ,

I~

150

//

I00 A 50 40 30 20

I0

t

2

J_

4

t

6 Time

t

B

I0

f

(h)

Fig. 4- Growth curves ok 5. aureus on casein hydrolyzate (see METHODS) in the presence of DLF (ioo ~g/ml) (O--O) and in the absence of DLF ( © - - O ) .

All the samples gave a continuous growth on solid rich medium after 6 and 24 h incubation. A culture of E. coli treated with 50/~g/ml D L F did not grow for 6 h, but at the end of 24 h it reached an A similar to that of the control culture. IOO #g/ml inhibited growth completely during the 24 h. Samples of cultures treated with the effective concentrations of D L F taken after 6 h incubation grew as single colonies on solid rich medium. Samples taken after 24 h incubation grew as a continuous growth at 50/~g/ml while the IOO #g/ml concentration sample was completely inhibited. DISCUSSION

D L F was isolated and purified b y successive application of trichloroacetic acid and salt precipitation. The progress of purification could be followed by assaying the fractions for the hemolytic activity. The final product was found to be about twice as active in this respect as the material isolated previously by paper electrophoresis. In Fig. 3 the hemolysis after 2 h is plotted against protein concentration. The ratio between the two slopes indicates the extent of purification. This result is in keeping with the picture obtained on gel electrophoresis, where the material isolated from paper gives two major bands of about equal intensity. The results obtained for amino acid composition, terminal amino acids and the Biochim. Bi ophy s . Acta, 151 (1968) 53-60

(io

~. ALOOIr-HIRSCH, A. I)E VRIES, A. BERGER

determination of molecular weight by physical methods show that D L F is a single chain protein of very small molecular size, approx. 6o amino acid residues. The presence of 8 half-cysteine residues (none of them in the reduced state in the native molecule) forming disulfide linkages indicates a high degree of intramolecular erosslinking. The amino acid composition is a rather unusual one. The protein is highly basic : 12 of the residues (2o%) are basic ones; the maiority of the 7 acidic residues seem to be in the amide form. Another unusual feature of the molecule is that it contains single residues per molecule of 6 amino acids: histidine, arginine, glutamic acid, alanine, tyrosine and phenylalanine. Remarkable too is its high content in branched-chain aliphatic amino acids (I2 residues, 2o%) and its proline content (5 residues, 8%) is unusually high. On the basis of its composition one might assume that D L F acts as a cationic detergent. This picture is supported by the observation that D L F resembles synthetic polypeptides composed of hydrophobic amino acids (leucine) and basic amino acids (ornithine) in that it also causes inhibition of growth in some bacteria. In this case, it had been shown that the above polypeptides cause damage to the bacterial cell membrane 2°. The analogy seems to support the proposed mode of action of D L F on red cells. Recently the structure of the basic polypeptide melittin ~1, isolated from bee venom, has been described22, 2a which resembles in its activity the snake-venom DLF. It has about half the molecular weight of cobra-DLF and a quite different amino acid composition, resembling it, however, in the content of basic amino acids (2o%) and branched-chain amino acids (35 %). Its hemolytic action is ascribed to its detergentlike effect 2a. REFERENCES K. SLOTTA, Progr. Chem. Org. Nat. Prod., 12 (1955) 4 °6. E. HABERMANN AND W. NEUMANN, Arch. Exptl. Pathol. Pharmakol., 223 (I954) 388. E. CONDREA, A. DE VRIES AND J. MAGER, Biochim. Biophys. Acta, 84 (1964) 60. CH. KIRSCHMANN, E. CONDREA, N. MOAV, S. ALOOF AND A. DE VRIES, Arch. Intern. Pharmacodyn., 15o (1964) 372. 5 E. CONDREA, W. AvI-DoR AND J. MAGER, Biochim. Biophys. Acta, t l o (1965) 337. 6 E. CONDREA, Z. MAMMON, S. ALOOF AND A. DE VRIES, Biochim. Biophys. Acta, 84 (1964) 365 . 7 E. CONDREA, J. KENDZERSKY AND A. DE VRIES, Experientia, 21 (1965) 461. 8 R. A. REISFELD, V. J. L E w i s AND D. E. WILLIAMS, Nature, 195 (1962) 281. 9 0 . H. LowRY, N. J. ROSEBROUGH, A . L . FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. IO G. I(EGELES, J. Am. Chem. Soc., 74 (1952) 5532. 11 D. A. YPHANTIS, Ann. N . Y . Acad. Sci., 88 (196o) 586. 12 J. R. SPIES AND D. C. CHAMBERS, Anal. Chem., 21 (1949) 1249. 13 K. I{ANDERATH, Thin Layer Chromatography, Academic Press, N e w York, 1963, p. 98. 14 Y. BURSTEIN AND A. I'ATCHORNIK, Israel J. Chem., 3 (1966) 9715 G. R. STARK AND D. G. SMYTH, J. Biol. Chem., 238 (1963) 214. 16 G. V. MARINETTI, M. ALBRECHT, T. FORD AND E. STOTZ, Biochim. Biophys. Acta, 36 (1959) 4. 17 C. F. REED, S. V. S~VISHER, G. V. MARINETTI AND E. G. EDEN, J. Lab. Clin. Med., 56 (196o) 281. 18 B. D. DAVIS AND E. S. MINGOLI, J. Bacteriol., 60 (195 o) 17. 19 E. KATCHALSKI, L. BICHOWSKI-SLOMNICKI AND B. E. VOLCANI, Bioehem. J., 55 (1953) 671. 20 L. BICHOWSKY-SLOMNICKI, A. BERGER, J. KURTZ AND E. KATCHALSKI, Arch. Biochem. Biophys., 65 (1956) 400. 21 VV. NEUMANN, E. HABERMANN AND U. NANSEN, Arch. Exptl. Pathol. Pharmakol., 217 (1953) 13o. 22 E. FIABERMANN AND J. JENTSCH, Z. Phys. Chem., 348 (1967) 37. 23 G. KREIL, Monatsh. 96 (1965) 2o61. 24 P. D. BOYER, J. Am. Chem. Soc., 76 (1954) 4331I 2 3 4

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