Studies on ε-prototoxin of Clostridium perfringens type D physicochemical and chemical properties of ε-prototoxin

Biochimica et Biophysica Acta, 412 (1975) 62-69

© Elsevier ScientificPublishing Company, Amsterdam - - Printed in The Netherlands BBA 37197 STUDIES ON e-PROTOTOXIN OF C L O S T R I D I U M P E R F R I N G E N S TYPE D P H Y S I C O C H E M I C A L A N D C H E M I C A L PROPERTIES OF e-PROTOTOXIN

A. F. S. A.

HABEEB

Division of Clinical Immunology and Rheumato!ogy, Department of Medicine and Departments of Microbiology and Biochemistry, The University of Alabama School of Medicine, Birmingham, Ala. 35294 (U.S.A.)

(Received March 17th, 1975)

SUMMARY e-Prototoxin of clostridium perfringens type D consists of one polypeptide chain of 311 amino acids with the following composition: Asps2 Thr31 Ser2s Glu2s Prolz Gly17 Ala~4 Val2a Met5 Ile15 Leu~8 Tyro7 Phea Lysa~ His~ Args Try~. It has no free cysteine but contains one blocked cysteine. The N-terminal as well as the Cterminal residue is lysine. The ultracentrifuge pattern gave one single boundary having sz°0,w= 2.15 S and Dz°0,w= 5.56.10 -7 cm2/s. Calculation of the molecular weight from D2o,~,°and s °20,wgave a value of 34 250. The molecular weight determined from sedimentation equilibrium using ultraviolet optics gave a value of 33 000 -+- 1000. On the other hand molecular weights calculated from a calibrated Sephadex G-100 column in borate buffer was 25 000 and that in sodium phosphate, ionic strength 0.2, was 27 500. This discrepancy between values obtained in the ultracentrifuge and by gel filtration is attributed to adsorption of e-prototoxin by Sephadex.

INTRODUCTION Clostridium perfringens type D produces several exoantigens; the principal one is e-prototoxin which exists in an inactive form. It is activated by short exposure to trypsin to yield a powerful e-toxin, e-Prototoxin occurs in multiple forms which differ in their electrophoretic mobility but are immunochemically indistinguishable [1 ]. These multiple forms varied in their activation ratios and specific activities [1]. By fractionating crude e-prototoxin on DEAE-cellulose followed by CM-cellulose, a major constituent (e-prototoxin) designated F1,3 was obtained which showed a specific activity of 3. l06 to 4.10 6 m o u s e MLD/mg protein. Physicochemical and chemical properties of e-prototoxin of different purity were reported by various investigators. Orlans et al. [2] separated e-prototoxin on DEAE-cellulose and found its sedimentation coefficient to be 2.8 S and its molecular weight 38 000 ~ 5000. Thomson [3, 4] found that crystalline e-prototoxin sedimented at 2.48 S and its molecular weight was 40 500. Habeeb [5] found the s20,~, of e-prototoxin separated by DEAE-cellulose chromatography to be 2.85 S and the molecular weight to be 23 200-25 000.

63 e-Prototoxin separated from DEAE-cellulose was found to yield a major fraction with the highest specific activity so far reported when rechromatographed on CM-cellulose [1 ]. The purpose of this investigation is to determine some chemical and physicochemical properties of e-prototoxin. A preliminary report on this work has appeared [6]. MATERIALS AND METHODS

Materials. Cloning, culture of Clo. perfringens type D, preparation of crude e-prototoxin and chromatography on DEAE-cellulose and CM-cellulose was as described previously in detail [1]. Pure e-prototoxin was fraction F1,3 from CMcellulose chromatography and will be referred to as e-prototoxin. Carboxypeptidase A and carboxypeptidase B were obtained from Worthington Biochemical Corporation. Amino acid analyses. Samples of e-prototoxin from two batches were hydrolyzed with constant-boiling HC1 in sealed evacuated tubes for 20 and 72 h. Amino acid analyses were done on a Beckman Model 120C automatic amino acid analyzer. Tryptophan was determined by reaction with O-nitrobenzene sulfenyl chloride in 99 ~o formic acid for 3 h [7]. A sample of reduced and carboxymethylated e-prototoxin was hydrolyzed for 24 h and S-carboxymethyl cysteine was determined on the amino acid analyzer. Determination of free sulfhydrylgroups. To 0.02/zmol of e-prototoxin in 1 ml of 8 M urea was added 5 ml of sodium dodecyl sulfate/sodium phosphate buffer (2 ~o sodium dodecyl sulfate in 0.08 M sodium phosphate buffer, pH 8, containing 0.5 mg EDTA/ml). The sulfhydryl groups were quantitated by Ellman's reagent [8] Nbs2 (5,5'-dithio-bis(2-nitrobenzoic acid)) as described previously [9, I0]. Determination of free sulfhydryl groups. To 0.02/~mol of e-prototoxin in 1 ml of fl-mercaptoethanol in Tris/glycine buffer, pH 7, containing 0.5 mg EDTA/ml as described previously [10, 11]. The reduction was done in the absence as well as in the presence of various concentrations of guanidine. After reduction the sulfhydryl groups were quantitated by Nbsz and calculated per tool of e-prototoxin (molecular weight 33 000). Reduction and carboxymethylation ofe-prototoxin. To 50 mg e-prototoxin dissolved in 4 ml Tris/glycine buffer, pH 7.0, containing 0.5 mg EDTA/ml, was added 1 ml 0.25 M fl-mercaptoethanol. The protein was allowed to react for 1 h then alkylated with 1.6 molar excess of iodoacetamide at pH 8 for 15 min. The completeness of the reaction was tested with Nbsz. After alkylation the solution was dialyzed against water and freeze-dried. Determination of N-terminal residue in e-prototoxin, e-Prototoxin was allowed to react in 66 ~ ethanol with l-fluoro-2,4-dinitrobenzene for 2.5 h. The dinitrophenyl (Dnp) prototoxin was precipitated, washed with water, ethanol and ether and hydrolyzed with 6 M HC1 for 12 h. The N-terminal Dnp amino acid was extracted with ether and subjected to two-dimensional paper chromatography on Whatman No. 1 using an ethylbenzene system [12] in the first direction followed by phosphate buffer in the second direction [13]. The colored spots were cut out, eluted with 0.1 M NaHCO3 and the absorbance determined at 360 nm. Quantitation was performed by use of appropriate factors obtained by subjecting a mixture of known amino acids to reaction, extraction and chromatography as described previously [12].

64

Determination of C-terminal amino acid in e-prototoxin, e-Prototoxin (2 mg/0.5 ml) in 0.1 M triethylamine, p H 8.2, was allowed to react with 0.1 ml carboxypeptidase A for 2 h (enzyme :protein ratio 1:50) at 40 °C. The reaction was stopped by addition of 0.1 ml M HC1. Aliquots of the solution were subjected to amino acid analysis. A sample of e-prototoxin was also digested with carboxypeptidase B. Determination of Stokes radius and diffusion coefficient. A 1.5 × 120 cm column was packed with Sephadex G-100 in borate buffer p H 8 (0.05 M in borate and 0.05 M KC1) and also in sodium phosphate, p H 7.5 and ionic strength of 0.2. The column was calibrated with bovine serum albumin, ovalbumin, soybean trypsin inhibitor and dextran blue to determine the effective pore radius as described previously [14]. D a t a f r o m Table I I I of Ackers [15] was used to calculate the Stokes radius of e-prototoxin f r o m its elution volume and the determined effective pore radius of the gel in the calibrated column. Determination of the sedimentation and diffusion coefficients, e-Prototoxin was dissolved and dialyzed against sodium phosphate, p H 7.5, and ionic strength 0.1. Sedimentation experiments were performed at three protein concentrations (3.23, 4.85 and 6.46 mg/ml) at 59 780 rev./min in a Model E ultracentrifuge at 20 °C using a synthetic b o u n d a r y cell. Measurements on the pattern were made with a N i k o n microcomparator. Sedimentation rates were calculated and corrected to standard conditions (s20,w) as described by Schachman [16]. The diffusion experiments were performed in the analytical ultracentrifuge using a double sector capillary tube synthetic b o u n d a r y cell and Schlieren optics. TABLE I AMINO ACID COMPOSITION OF e-PROTOTOXIN Amino acid

Residues per mol (33 000) No. 37 No. 34 No. 34 (20 h) (20 h) (72 h)

S-Carboxymethyl cysteine Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe Lys His Arg Try

0.9 50.1 28.8 22.7 26.7 11.8 16.7 14.1 25.0 5.3 13.8 18.3 16.7 8.4 30.4 2.1 5.1

0.93 50.8 30.0 23.7 28.0 11.8 17.3 14.5 26.0 5.4 14.1 18.1 17.0 8.4 30.1 2.2 5.0

0.9 51.6 26.7 20.3 27.9 11.9 16.6 13.6 28 5.2 15.4 18.2 16.7 8.3 31.1 1.9 4.8

Residue to nearest integer

~ Amino acid This work

Ref. 4

1 52 31" 25* 28 12 17 14 28 5 15 18 17 8 31 2 5 2**

0.41 17.0 8.88 6.25 10.3 3.1 2.73 2.84 7.91 2.09 4.96 5.87 7.81 3.52 11.25 1.09 2.49 1.49

0.59 17.3 10.00 6.07 9.95 8.8 8.07 3.00 6.95 1.92 3.72 5.8 6.02 1.72 14.2 1.28 2.55 0.58

* Values corrected for losses during hydrolysis by extrapolation to zero time of hydrolysis. ** Determined by o-nitrophenyl sulfenyl chloride.

65 The centrifuge was operated at 3617 rev./min and 20 °C. Diffusion coefficients were calculated by the maximum "ordinate-area" method of Ehrenberg [17] and corrected to standard conditions (D20,~,) as described by Schachman [16]. Determination of molecular weight of e-prototoxin. (a) Molecular weight was determined by sedimentation equilibrium in a Beckman Model E ultracentrifuge. The light source monochromator was set at 280 nm and the temperature was regulated to 20 °C. The sample column height was 3 mm. The protein was dialyzed against sodium phosphate buffer, ionic strength 0.2 and p H 7.5. The partial specific volume of the prototoxin was calculated from its amino acid composition [18] and found to be 0.727 ml/g. Molecular weight values were calculated from the slope of the absorbance plots according to the general procedures described by Chervenka [19]. (b) The molecular weight of e-prototoxin was calculated from s2°0,wand the diffusion coefficient using the Svedberg equation [20]. (c) The molecular weight was also derived from the elution volume on a calibrated Sephadex G-100 column using a plot of the elution volume vs log molecular weight of proteins of known molecular weights (bovine serum albumin, ovalbumin and soybean trypsin inhibitor). RESULTS Table I shows the amino acid analysis of two different batches of e-prototoxin after 20 and 72 h of digestion and the number of residues to nearest integer per mol of e-prototoxin of molecular weight 33 000. The values were converted in terms of percent amino acid composition and compared with values obtained by Thomson [4]. Generally there is some agreement in some of the amino acids. However great discrepancy occurs in the values of proline, glycine and phenylalamine. Moderate differences are evident in threonine, valine, isoleucine, tyrosine and lysine. Table II reports on the cysteine and cystine content. It shows the absence of free cysteine. However, there is one mol of cysteine per mol of protein which is blocked and is reduced with fl-mercaptoethanol in absence of guanidine. Denaturation

TABLE II CYSTEINE AND CYSTINE CONTENT OF e-PROTOTOXIN Order of 1 2 3 4 5 6 7 8 9 10 11

Treatment

mol -SH/mol protein

Free sulfhydryl Reduction with 0.05 M fl-mercaptoethanol in 4 M guanidine 1 h As above 3 h As above 6 h Reduction with 0.05 M fl-mereaptoethanol in absence of guanidine for 3 h In presence of 0.85 M guanidine In presence of 1.7 M guanidine In presence of 2.55 M guanidine In presence of 3.4 M guanidine In presence of 4.25 M guanidine In presence of 5.18 M guanidine

0 1.1 1,07 1.02 0.9 0.85 1.09 1.10 1.05 1.09 1.0

66 TABLE III N- AND C-THERMINAL AMINO ACIDS IN e-PROTOTOXIN Amino acid

mol amino acid/mol protein M~ 33 000

N-terminal amino acid

0.78 lysine as di-DNP-lysine

C-terminal amino acid in: (a) Carboxypeptidase A (b) Carboxypeptidase B

0 1.02 lysine

o f e - p r o t o t o x i n with guanidine up to 5.1 M c o n c e n t r a t i o n does n o t affect the liberation o f cysteine. The N - t e r m i n a l a n d the C-terminal a m i n o acids are r e p o r t e d in Table III. The results indicate t h a t the p r o t e i n is f o r m e d f r o m one p o l y p e p t i d e chain with an N - t e r m i n a l lysine a n d C-terminal lysine. The absence o f o t h e r a m i n o acids is evidence for the chemical h o m o g e n e i t y o f e-prototoxin. Physicochemical properties, e - P r o t o t o x i n sediments as a single b o u n d a r y in s o d i u m p h o s p h a t e buffer indicating a h o m o g e n e o u s p r e p a r a t i o n (Fig. 1). The sedim e n t a t i o n coefficient s 21),',v o = 2.15 S was c o n c e n t r a t i o n - i n d e p e n d e n t within the range e x a m i n e d (3.24-6.46 mg/ml). The diffusion coefficient D2°0.w was 5.56. l0 -7 cm2/s. The m o l e c u l a r weight calculated f r o m b o t h the s e d i m e n t a t i o n a n d diffusion coefficients was 34 250. Results o b t a i n e d f r o m s e d i m e n t a t i o n equilibrium at 12 590 a n d 15,220 rev./min with various concentrations are shown in Table IV. The average m o l e c u l a r weight calculated is 33 000 ~: 1000. A p l o t o f log _4 as a function o f r E for three c o n c e n t r a t i o n s : 0.5, 0.61 a n d 0.82 a b s o r b a n c e units at 280 n m is shown in

Fig. 1. Ultlacentrifugal patterns of e-prototoxin. Photographs were taken at the following times in minutes after acceleration to 59 780 rev./min.: A, 0; B, 16; C, 32; D, 48; and E, 64. TABLE IV MOLECULAR WEIGHT OF e-PROTOTOXIN FROM SEDIMENTATION EQUILIBRIUM Absorbance of e-prototoxin

Rev./min.

Mol. Wt

Rev./min.

Mol. wt

0.50

12 590

15 220

0.61 0.82

12 590 12 590

32 400 32 800 33 200 34 900 34 200

31 800 32 600 31 900 33 800 32 200

67 08

A

0.6 0.4

02

O~

~ 0

LO 0.8 06

~

0.4

N

B

7

rn 0.2 rr" 0 ch rn < 0.1

2.0

I0 0.8 06 04

0.2

J 47

48

49

50

51

r 2 (cm 2)

Fig. 2. Distribution of e-prototoxin at sedimentation equilibrium in sodium phosphate buffer, pH 7.5, ionic strength 0.2. The initial concentrations of the samples were: (A), 0.50; (B), 0.61; and (C), 0.82 absorbance units at 280nm. Each sample was examined at two rotor speeds: (Q--~), 12 590 rev./min, and ((3--©), 15 220 rev./min. Apparent molecular weights are given in Table IV. Figs 2A, B and C at two different speeds: 12 590 and 15 220 rev./min. A slight curvature in some of the plots indicates a small degree of heterogeneity in the preparation. However, comparison of the molecular weight values for these samples at each of the rotor speeds provided additional evidence that the heterogeneity was minimal. Gelfiltration. Determination of the Stokes radius of e-prototoxin on a calibrated Sephadex G-100 column gave a value of 1.96 nm. The value of the diffusion coefficient calculated from the Stokes radius was 10.6-10 -7 cm2/s. The molecular weight calculated from sedimentation coefficient and diffusion coefficient (calculated from the Stokes radius) was 23 000 and that obtained from the elution volume on calibrated Sephadex G-100 column was 25 000 in borate buffer and 27 500 in sodium phosphate of ionic strength 0.2. DISCUSSION e-Prototoxin is the major exoantigen of Clo. perfringens type D. It was separated and characterized by several investigators but there is lack of agreement on the

68 molecular weight or amino acid composition. This may be due to the impurity of the isolated e-prototoxin, e-Prototoxin is present in the culture fluid in a nontoxic form which becomes activated by brief proteolytic digestion. The activation ratio, which is the ratio of toxicity after enzyme activation to that before treatment, is a measure of the presence of e-toxin in e-prototoxin preparations. A low activation ratio is associated with the presence of large proportions of e-toxin. The preparation of Orlans et al. [2] was mostly e-toxin shown by an activation ratio of 2. It had a specific activity after activation of 44 800 mouse MLD/mg protein. Thomson's crystalline eprototoxin [3, 4] showed that 92.5 ~ of the protein had a molecular weight of 40 500 and that the molecular weight of the contaminating material did not exceed 14 000. The specific activity was about 3.5.10 ~ mouse MLD/mg protein and it had an activation ratio of 5~30. A highly purified preparation of e-prototoxin was prepared from Clo. perfringens type D grown on a protein-free medium [1 ] (to prevent contamination with protein from the medium). It was found to be immunochemically homogeneous and had the highest specific activity and activation ratio so far reported [1 ] (specific activity 3.3.106 to 4.2.106 mouse MLD/mg protein and activation ratio 1750-3300). The work shows that e-prototoxin consists of one polypeptide chain formed of 311 amino acid residues with one N-terminal lysine and one C-terminal lysine. The absence of any C-terminal amino acid revealed by carboxypeptidase A and the appearance of only lysine with carboxypeptidase B is evidence of its chemical homogeneity. It has no free cysteine but one blocked cysteine residue which is available to reduction with fl-mercaptoethanol in aqueous buffer. The amino acid composition is at variance with that reported by Thomson [3] especially in proline, glycine and phenylalanine. It is also different from that in a previous report [5] where the presence of hydroxyproline and hydroxylysine was observed. This discrepancy could be due to presence of protein contaminants from the culture medium. These have been completely eliminated by growing the bacteria in protein-free medium. The molecular weight obtained by sedimentation equilibrium, 33 000 ± 1000 is in good agreement with a value 34 250 calculated from the sedimentation and diffusion coefficients. The plot of log A as a function of r 2 provided evidence that the heterogeneity was minimal. However the molecular weight calculated from the elution volume by gel filtration on Sephadex G-100 was lower, 25 000 and 27 500 when the column was run in borate buffer and phosphate buffer respectively. Anomalous results were obtained with lysozyme. Elution was retarded giving an apparent molecular weight of 7700 on Sephadex G-100 [21]. Aromatic amino acids were shown to be retarded on Sephadex [22] and a protein rich in these amino acids may be expected to behave anomalously, e-Prototoxin contains 17 tyrosines, 8 phenylalanines and 2 tryptophans, which may account for its delayed elution. It is not known whether the ability of e-prototoxin to bind to polysaccharides may contribute to its toxicity since e-toxin shows similar binding tendencies. It may be relevant to indicate that the activation of e-prototoxin to toxin is accompanied by very little change in molecular weight. Although the activation is accompanied by conformational changes [23], immunochemical reactivity is unchanged. ACKNOWLEDGEMENTS I wish to thank Dr Ralph E. Schrohenloher for the sedimentation results.

69 REFERENCES 1 Habeeb, A. F. S. A. (1969) Arch. Biochem. Biophys. 130, 430-440 20rlans, E. S., Richards, C. B. and Jones, V. E. (1960) Immunology 3, 28-44 3 Thomson, R. O. (1962) Nature 193, 69-70 4 Thomson, R. O. (1963) J. Gen. Microbiol. 31, 79-90 5 Habeeb, A. F. S. A. (1964) Can. J. Biochem. 42, 545-554 6 Habeeb, A. F. S. A. (1972) Fed. Proc. 31, 4083 7 Scoffone, E., Fontana, A. and Rocchi, R. (1968) Biochemistry 7, 971-979 8 Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77 9 Habeeb, A. F. S. A. (1966) Biochim. Biophys. Acta 115, 440-454 10 Habeeb, A. F. S. A. (1972) Methods in Enzymology Vol. 25, pp. 457-464, Academic Press, New York 11 Habeeb, A. F. S. A. (1967) Arch. Biochem. Biophys. 121, 652-664 12 Habeeb, A. F. S. A. (1958) J. Pharm. Pharmacol. 10, 591-592 13 Levy, A. L. (1954) Nature 174, 126-127 14 Habeeb, A. F. S. A. (1966) Biochim. Biophys. Aeta 121, 21-25 15 Ackels, G. K. (1964) Biochemistry 3, 723-730 16 Schachman, H. K. (1957) Methods in Enzymology, Vol. 4, pp. 32-103, Academic Press, New York 17 Ehrenberg, A. (1957) Acta Chem. Scand. 11, 1257-1270 18 Cohn, E. J. and Esdall, J. T. (1943) in Proteins, Amino Acids and Peptides, pp. 370-381, Rheinhold, New York 19 Chervenka, C. H. (1969) in A Manual of Methods for the Analytical Ultracentrifuge, pp. 39-67 Beckman Instruments, Inc., U.S.A. 20 Svedberg, T. and Pederson, K. O. (1940) The Ultracentrifuge, pp. 3-15, Oxford Unive.rsity Press, London 21 Whitaker, J. R. (1963) Anal. Chem. 35 1950-1953 22 Porath, J. (1960) Biochim. Biophys. Acta 39, 193-207 23 Habeeb, A. F. S. A., Lee, C.-L. and Atassi, M. Z. (1973) Biochim. Biophys. Acta 322, 245-250