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UBC Theses and Dissertations

Changes in toxicity of Clostridium Botulinum type E toxin by chemical modification and enzymatic cleavage Ko, Arthur S.C. 1965

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CHANGES IN TOXICITY OP CLOSTRIDIUM BOTULINUM TYPE E TOXIN BY CHEMICAL MODIFICATION AND ENZYMATIC CLEAVAGE by ARTHUR S.C. KO B.Sc. University of B r i t i s h Columbia 1 9 6 3 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Bacteriology and Immunology We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1 9 6 5 In presenting th i s thes i s in p a r t i a l f u l f i lmen t of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f r ee l y a va i l ab l e fo r reference and study. I fur ther agree that per-mission for extensive copying of t h i s thes i s for s cho la r l y purposes may be granted by the Head of my Department or by h i s representatives,, It i s understood that copying or p u b l i -ca t ion of th i s thes i s for f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion. Department of The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada 2£ o^e. /9£f i ABSTRACT The single residue of cysteine i n C l . botulinmn type E s t r a i n Iwanai toxin has been linked with t o x i c i t y , by chemical modification using p-chloromercuribenzoate. A peptide containing the oysteine residue has been isolated by exhaustive t r y p t i c digestion of the toxin molecule tagged with N-(4-dimethylamino-3,5-dinitrophenyl) maleimide, and subsequent g e l f i l t r a t i o n with Sephadex G-25 and descending paper chromatography. The toxic peptide of trypsin-activated toxin was isolated by fractionation through a composite Sephadex G--75 and G-50 column. By chymotryptic and t r y p t i c digestion of the toxin at pH 5.8, a toxic fragment has been isolated by gel f i l t r a t i o n with Sephadex G--25. On the basis of quantitative amino acid analyses, the molecular weights of the intact toxin, the trypsin-activated toxin and the chymotrypsin-"trypsin fragmented toxin have been estimated to be 14,000-16,000, 10,000-12,000 and 4,000-6,000 respectively. Although the mechanism of t r y p t i c a c t i v a t i o n was found to involve c h i e f l y the removal of at least 18 amino acid residues from the N-terminus of the toxin molecule, the manner of reduction by cleavage has not been determined for the chymotrypsin-trypsin fragmented toxin. i i TABLE OF CONTENTS Introduction . l a Review of Literature 1 I. Chemical modification .1 A. Introductory remarks 1 B. Chemical modification of botulinus toxins 2 C. Methods of chemical modifications 4 D. Modification of -SH groups 6 I I . Enzymatic cleavage 10 A. Introductory remarks 10 B. Peptic digestion 11 C. Tryptic digestion .12 D. Chymptryptic digestion 13 Materials and Methods 14 Culture 14 Toxin production 14 Treatment of d i a l y s i s tubing 14 Quantitative assay 15 Potency assay 15 Acid hydrolysis procedure 15 Perform!c acid oxidation..... 16 Amino acid analysis ...16 N-terminal amino acid determination 16 Inactivation by iodoacetate 17 Inactivation by PCMB 17 Chromat ographi c t e chni que s 18 Preparative paper chromatographic technique 20 E l u t i o n from chromatographic paper ...21 I s o l a t i o n of a DDPS-peptide 21 Trypsin a c t i v a t i o n 22 Chymotrypsin-trypsin degradation. 23 i i i I s o l a t i o n of the trypsin-activated toxin 23 Is o l a t i o n of the chymotrypsin-trypsin fragmented toxin .24 Results and Discussion 25 Quantitative assay 25 Inactivation by iodoacetate 29 Inactivation by PCMB 29 I s o l a t i o n of a DDPS-peptide 33 Tryptic a c t i v a t i o n 38 Chymotrypsin-trypsin fragmentation. 44 General Discussion and Conclusions 50 Bibliography i v LIST O P FIGURES AND TABLES Figure 1 Quantitative assay of toxin.... 27 Figure 2a Fractionation of the once-trypsinized DDPM-labelled toxin by Sephadex 0-25 34 Figure 2b Fractionation of the twice-trypsiniaed DDPM-labelled toxin by Sephadex G-25 35 Figure 3a E f f e c t of pH and time on the t r y p t i c digestion of type E toxin ..39 Figure 3b Fractionation of the trypsin-activated toxin by Sephadex G-75 and G-50 41 Figure 4a Eff e c t of chymotryptic-tryptic digestion on the t o x i c i t y of type E toxin at pH 5.8...45 Figure 4b Fractionation of the chymotrypsin-trypsin fragmented toxin by Sephadex G-25 47 Table 1 Reaction of toxin with PCMB at pH 7.0 30 Table 2 Type E toxin, trypsin-activated toxin and chymotrypsin-trypsin fragmented toxin compared i n terms of amino acid contents, N-terminal residues and comparative t o x i c i t i e s 49 V ACKNOWLEDGEMENTS I wish to acknowledge the help generously given by both Dr. G.Dixon (Department of Biochemistry, U.B.C.) and Dr. J.T. Tremaine (Federal Department of Agriculture Research Station, Vancouver) who helped with the amino acid analyses and graciously gave t h e i r time f o r consul-tat i o n and advice. I also wish to thank my supervisor, Dr. J . Gerwing, whose patience and understanding, nearly, but never once faltered, even at times of extreme exasperation. With-out her constant encouragement, able supervision and effervescent nature t h i s study would not have been possible. INTRODUCTION C l . botulinum type E produces a toxic protein which can be obtained i n a highly p u r i f i e d state and i t s mol-ecular weight has been determined. It i s s e r o l o g i c a l l y d i s t i n c t from other types of botulinus toxins, which i n -dicate differences i n t h e i r molecular structures. However, they share a common mode of action which allegedly involves the i n h i b i t i o n of acetylcholine release mechanism at nerve terminals. Exactly how the toxin molecule participates i n the inte r a c t i o n i s unknown. The common mode of action of the various types of botulinus toxins suggests the possible existence of a common, b i o l o g i c a l l y active portion i n the toxin molecules. This investigation attempts to characterise the b i o l o g i c a l l y active portion of type E toxin. Attempts were made to determine which reactive side chains of the amino acid residues i n the toxin molecule are associated with bio-l o g i c a l a c t i v i t y , ( i e . which amino acid residues are i n -volved i n the active s i t e ) , by means of chemical modification. Having established the above, attempts were then made to iso l a t e a r e l a t i v e l y small peptide from that predetermined region of the toxin molecule. Previous studies have suggested that the phenomenon of t r y p t i c a c t i v a t i o n of type E toxin i s due to molecular fragmentation of the toxin molecule by trypsin giving r i s e to a smaller, more active peptide. Attempts were made to confirm the above hypothesis. The l a t t e r ' s confirmation led to further attempts of frag-mentation by means of other proteolytic enzymes and the l b subsequent i s o l a t i o n of a low molecular weight, toxic fragment (peptide) from type E toxin. The structure of such a toxic peptide may be characterised with r e l a t i v e ease, and i n turn may y i e l d the key to the understanding of the t o x i c i t y of botulinus toxins. S p e c i f i c methods available f o r the chemical modific-ation of the reactive side chains of amino acid residues are l i m i t e d . Therefore, either the single h i s t i d i n e or cysteine residue i n the toxin molecule, as revealed by quantitative amino acid analysis, became the obvious target for chemical modification studies. Moreover, the observations that pretreatment of the d i a l y s i s tubing with versene prevents the in a c t i v a t i o n of the toxin during the process of d i a l y s i s , and mercaptoethanol s t a b i l i z e s the toxin, strongly suggest that the single cysteine residue was the obvious choice f o r chemical modification* Because the natural route of toxigenesis i s per oe. the toxin molecules must be somewhat resistant to proteolytic enzymes encountered i n the digestive t r a c t . In other words, even though the toxin molecule has been reduoed i n molecular size through cleavage by proteolytic enzymes, the diminished fragment must remain b i o l o g i c a l l y active f o r i n t e s t i n a l absorption. Hence, the proteolytic enzymes were the frag-mentation agents of choice f o r the toxin molecule. REVIEW OF LITERATURE j c I. CHEMICAL MODIFICATION A. Introductory remarks Botulinus toxins may be considered as molecules con-s i s t i n g of single polypeptide chains due to the lack of contrary evidence. The length of the polypeptide chain and the primary structure vary among the types of botulinus toxins. The amount of cross-links and c o i l i n g i s dependent on the primary structure. Chemical bonds of varying strengths responsible f o r the secondary and t e r t i a r y struc-tures can be subjected to modification. Denaturation or i n a c t i v a t i o n may be defined as modification of any of these chemical bonds giving r i s e to a polypeptide chain which di f f e r e d i n stereochemical conformation from the b i o l o g i c -a l l y active molecule. The degree of denaturation can be assayed from the b i o l o g i c a l a c t i v i t y , and manipulated by chemical substitution or conversion of the diverse reactive side chains of the amino acid residues i n the polypeptide. In theory, i t i s possible to modify chemically any amino acid residue with a side chain exhibiting a d i s t i n c t i v e . type of r e a c t i v i t y ; but i n practice, only a few suitable chemicals are available and well established. During the recent years, a number of proteins have been successfully modified by chemical means that resulted i n t h e i r loss of b i o l o g i c a l a c t i v i t y . This has provided an approach for locating groups of amino acid residues i n proteins which may be referred to as the active centres ( s i t e s ) . 2 B. Chemical modification of botulinus toxins The lack of data on type £ toxin necessitates a review of the chemical studies undertaken on type A toxin. In order to understand what the chemical studies have revealed, the physical and chemical nature of type A toxin must be considered. Type A toxin can be obtained i n c r y s t a l l i n e form (Lamanna et a l . , 1946). The elemental and amino acid analyses as performed by Buehler et a l . , (1947) have shown i t to be a simple protein. I t has a calculated molecular weight of 900,000 on the basis of physical measurements (Putnam et a l . , 1948). Lamanna (1948) found that c r y s t a l -l i n e type A toxin has both toxic and hemagglutinating prop-erties and drew attention to the lack of i d e n t i t y between the toxic and hemagglutinating factors (Lamanna and Lowen-th a l , 1951; Lowenthal and Lamanna, 195l). The diss o c i a t i o n of c r y s t a l l i n e type A toxin into subunits under the proper conditions of pH and i o n i c strength has been demonstrated by Wagman and Bateman (l951» 1953) and by Wagman (1954). Moreover, the highly p u r i f i e d type A toxin as isolated by G-erwing et a l . , (1965) has a molecular weight of 12,200 as calculated on the basis of physical measurements served to point out emphatically that the heterogeneity of c r y s t a l -l i n e type A toxin i s highly probable. In spite of the heterogeneous nature of the c r y s t a l -l i n e type A toxin, various investigators have conducted chemical and physical studies on i t . When Boroff (1959) found experimentally that d e t o x i f i c a t i o n was coincidentally 3 accompanied by the lose of fluorescence, he postulated that t o x i c i t y was linked with fluorescence i n the toxin molecule. The above hypothesis was invalidated by Schantz et a l . , (l96l) who demonstrated that the fluorescence was retained when the toxin molecule was detoxified by 614 urea. Weil et a l . , (1957) found that t o x i c i t y was destroyed by photooxid-ation i n the presence of methylene blue. Having confirmed this using the same method, Boroff and DasGupta (1964) re-lated t o x i c i t y to the tryptophan residues i n the toxin mol-ecule. More recently, Boroff and DasGupta (1965) used 2-hydroxy-5-nitrobenzyl bromide (HNBB), a "more or l e s s " spec-i f i c reagent for tryptophan residues and have claimed that t o x i c i t y i s d i r e c t l y linked to the tryptophan residues. Schantz and Spero (1957) reacted the toxin with ketene and found that t o x i c i t y was l o s t when the free -NHjj groups were reacted. They further claimed that the phenolic -OH and free -SH groups are not associated with t o x i c i t y . Spero and Schantz (1957) found that deamination of the toxin with nitrous acid caused rapid d e t o x i f i c a t i o n . Spero (1958) found that the decrease i n t o x i c i t y due to an imerease i n pH i s associated with the i o n i s a t i o n of a small number of epsilon -NH2 groups of the lysine residues. Thus the tox-i c i t y of botulinus toxins i s linked with the reactive side chains of tryptophan and lysine residues as revealed by chemical studies on type A c r y s t a l l i n e toxin. Since the l a t t e r has some 477 residues of ly s i n e and 82 residues of tryptophan (Buehler et a l . , 1947), and the chemical reagents 4 employed are not exclusively s p e c i f i c for reaction with lysine or tryptophan, the significance of the findings based on these chemical studies i s rather vague and dub-ious. Furthermore, studies r e l a t i n g molecular structure to b i o l o g i c a l a c t i v i t y can have fundamental significance only i f the material to be tested i s of a high degree of homogeneity. The b i o l o g i c a l evaluation of "impure* materials i s l i k e l y to y i e l d misleading r e s u l t s . C. Methods of chemical modification G-erwing et a l . , (1964) reported that type E toxin can be obtained i n a highly p u r i f i e d state and that the pretreatment of the d i a l y s i s tubing by versene prevents the i n a c t i v a t i o n of the toxin during the process of d i a l -y s i s . The l a t t e r fact hints at the p o s s i b i l i t y that the cysteine residues exist i n the toxin i n the reduced form. Further substantiation of such a p o s s i b i l i t y can be derived from the s t a b i l i s i n g e ffect of the toxin by mercaptoethanol which can be used to reduce or cleave the d i s u l f i d e bonds i n proteins (Thompson and O'Donnell, 1961). Fhotooxidation i n the presence of methylene blue (Weil et a l . , 1957) or a l k y l a t i o n by iodoacetate are the methods of choice f o r a preliminary study on the t o x i c i t y of type E toxin. Photo-oxidation i n the presence of methylene blue i s a pH-, and to a certain extent, temperature-dependent reaction, and w i l l modify tryptophan, h i s t i d i n e , methionine, tyrosine and cysteine residues without breaking the peptide bonds. 5 Because the reaction conditions i n photooxidation are d i f f i c u l t to control and the apparatus involved compli-cated, a l k y l a t i o n by iodoacetate was chosen i n t h i s study. Both iodoacetic acid and i t s derivative, iodoacetamide have been used successfully f o r the chemical modification of proteins. Goddard and Michaelis (1935) and Sela et a l . , (l959) have shown that a l k y l a t i o n by these reagents can be r e s t r i c t e d to -SH groups at a pH of about 8. Observed differences i n the r e a c t i v i t y between the two reagents are due to the negative charge on the iodoacetate ion. Yank-eelov and Eoshland (l96l) found that phosphoglucomutase reacts with one mole of iodoacetate with a 40$ loss of ac-t i v i t y but the action of iodoacetamide i s considerably more rapid. The mechanism of a l k y l a t i o n by iodoacetate i s best i l l u s t r a t e d with the various data obtained from i n a c t i v -ation of ribonuclease. Z i t t l e (1946) observed that ribo-nuclease could be slowly inactivated by iodoacetate and attributed the i n a c t i v a t i o n to the a l k y l a t i o n of -SH groups. Ribonuclease has been shown by Hirs et a l . , (1958) not to contain any free -SH groups. Thorough examination by Gund-lach et a l . , (1959) demonstrated that the a l k y l a t i o n of ribonuclease was a pH dependent reaction. Xn the absence of -SH groups and under well controlled pH, a l k y l a t i o n can be confined to the reactive side chains of certain amino acid residues; i e . at pH 5-6, the imidazole group of h i s -t i d i n e ; at pH 2-3, the thioether sulphur of methionine and at pH 8-10, the epsilon -NH2 group of lysine and the thioether 6 sulphur of methionine. Moore et a l . , (1953) investigated the d i f f e r e n t methods of reducing the d i s u l f i d e bonds i n proteins and examined the methods of converting the re-sultant cysteine residues to stable derivatives. They found that sodium borohydride was e f f e c t i v e i n the reduc-t i o n of ribonuclease and chymotrypsinogen; and iodoacetate was capable of forming the carboxymethylated product at pH 8.5. The completeness and the s p e c i f i c i t y of the re-action were checked by acid hydrolysis followed by quan-t i t a t i v e ion-exchange chromatography (Spackman et a l . , 1953). Botulinus toxins are inactivated at alkaline pHs, thus thwarting any attempt to evaluate precisely the i n -act i v a t i o n of -SH groups i n the toxin molecule by iodoacetate. D. Modification of -SH groups The importance of the role of sulphur i n proteins has been recognised and emphasised recently (Benesch et a l . , 1959; Boyer, 1959; C e c i l and McPhee, 1959; and C e c i l , 1963). Relatively l i t t l e i s known about the function of the methionine residues i n proteins. Recent studies with ribonuclease (Vithayathil and Richards, 1960a,b and 196la,b) and with myoglobin (Kendrew et a l . , 196l) sug-gested that the possible role of methionine i s stru c t u r a l ; i e . the formation of hydrophobic bonds. I t i s s u f f i c i e n t to say that the S-S group, when present, plays a major role i n s t a b i l i z i n g the three dimensional structures of proteins. The estimation of the number of -SH groups i n 7 the toxin molecule i s a prerequisite to the evaluation of the group's function. The formation of highly undissoc-iated mercaptides by s i l v e r s a l t s , mercuric s a l t s and org-anic mercury derivatives (RHgX) provides the basis for several estimation methods. S i l v e r s a l t s are stable, eas-i l y p u r i f i e d and t h e i r concentrations can be e a s i l y determined by amperometric t i t r a t i o n s (Benesch and Benesch, 1948; Ben-esch et a l . , 1955). However the resultant s i l v e r mercap-tides have a strong tendency to bind additional s i l v e r ions, thus often giving r i s e to misleading positive errors (Bur-ton, 1958). Mercuric ions are more s p e c i f i c than s i l v e r ions i n reaction with -SH groups and the complexes of mer-curic mercaptides with mercuric ions are less stable than the corresponding s i l v e r compounds ( C e c i l and McPhee, 1959). Simple t h i o l s react to form mercaptides of the type (RS) 2Hg, whereas proteins often give the half-mercaptides Prot.SHgX. In the reaction of mercuric ions and a given protein, the type of mercaptide formation w i l l depend on whether or not i t i s s t e r i c a l l y possible f o r two -SH groups i n the protein molecule to react with one mercuric ion. Denatured proteins normally form (Prot.S) 2Hg ( A l l i s o n and C e c i l , 1958). Org-anic mercury derivatives (RHgX) have a comparable s p e c i f i c -i t y f or -SH groups to that of mercuric ions and have the added advantage of being univalent. The R group can either be a l i p h a t i c or aromatic. The l a t t e r i s preferred as i t i s more soluble i n water and less poisonous. Among the mer-captide-forming reagents, organic mercury compounds and 8 notably £-chloromercuribenzoic acid (Hellerman, 1937; Hel-lerman et a l . , 1943) have long been the compounds of choice for the detection of -SH groups i n b i o l o g i c a l systems because of t h e i r s e l e c t i v i t y and the s t a b i l i t y of the r e s u l t i n g mer-captides . The use of these compounds as quantitative re-agents was re a l i s e d by Boyer (1954) who used the increase i n absorption that occurs at 250 mu when FCMB reacts with the -SH groups. The technique employed t i t r a t i o n of the mercurial with the -SH compound u n t i l there was no further change i n d i f f e r e n t i a l absorption. The method has the ad-vantage i n that the measurements indicate the amount of mer-captide formed rather than the uptake of the mercurial. Two types of -SH groups may be recognized from t h e i r r e a c t i v i t y toward the -SH reagents. The reactive -SH group has the same r e a c t i v i t y as do the simple t h i o l s . The unre-aotive -SH groups are those which show a low r e a c t i v i t y i n the native protein but normal r e a c t i v i t y a f t e r the protein has been denatured. The high r e a c t i v i t y of the reactive -SH groups has led investigators to postulate t h e i r part-i c i p a t i o n i n the active s i t e s of enzymes, and there are a large number of enzymes that can be inactivated by heavy metal reagents and reactivated by t h i o l s (Boyer, 1959). In very few cases, the -SH groups have been found to form part of the active s i t e . Since the introduction of Sanger's reagent for N-term-i n a l amino acid residue determination, the value of s p e c i f i c colored reagents i n protein chemistry has been recognised. 9 Witter and Tuppy (i960) established the s p e c i f i c i t y and reaction mechanism of the yellow -SH group reagent, N-(4-dimethylamino-3»5-dinitrophenyl)maleimide. Another reagent, N-(2,4-dinitroanilino)maleimide has been synthe-sised and was used by Clark-Walker and Robinson i n 1961. More recently, N-(4-dimethylamino-3,5-dinitrophenyl)mal-eimide has been used to l a b e l an essential -SH group of rabbit glyceraldehyde-3-phosphate dehydrogenase (Gold and Segal, 1964). In each case cited, the l a b e l l i n g with the colored -SH reagent led to successful i s o l a t i o n and sub-sequent characterisation of a small peptide. 1 0 I I . ENZYMATIC CLEAVAGE A. Introductory remarks Even though, chemical methods can provide an approach leading to the i s o l a t i o n of a r e l a t i v e l y small peptide from a b i o l o g i c a l l y active protein molecule, such a pep-tide may not constitute the entire active centre because the reactive side chains of amino acid residues which are adjacent i n sequence need not necessarily be adjacent ster-i c a l l y . Nevertheless, protein chemists to-day have recog-nised the relationship between b i o l o g i c a l a c t i v i t y and the reactive side chains of a few amino acid residues i n the protein molecule. Even i f the reactive side chain of a s p e c i f i c amino acid residue i n a given protein molecule having been chemically modified resulted i n the loss of b i o l o g i c a l a c t i v i t y , t h i s does not imply that the other amino acid residues i n the molecule are functionless. I t may be argued that only a certain number of these other amino acid residues are necessary f o r the maintenance of the stereochemical conformation of the molecule. How many and which amino acid residues i n a p a r t i c u l a r protein molecule f u l f i l l t h i s s t r u c t u r a l function must be known before such an argument can be entertained. Chemical meth-ods are available f o r s p e c i f i c fragmentation of protein molecules (Thompson, i 9 6 0 ) , but the loss of b i o l o g i c a l ac-t i v i t y may be due to the drastic reaction conditions rath-er than the fragmentation process i t s e l f . Thus, enzymatic degradation of protein molecules may provide the means of 11 y i e l d i n g a s m a l l , b i o l o g i c a l l y a c t i v e p e p t i d e . B. P e p t i c d i g e s t i o n Since the n a t u r a l route of t o x i g e n e s i s by b o t u l i n u s t o x i n s i s per os, the t o x i n molecule may be reduced i n molecular s i z e as i t passes down the d i g e s t i v e t r a c t . Moreover, t h i s s m a l l e r moiety must r e t a i n i t s t o x i c i t y f o r i n t e s t i n a l a b s o r p t i o n . The t o x i n molecule i s probab-l y s u bjected to s l i g h t d i g e s t i o n on encountering the gas-t r i c p r o t e o l y t i c enzyme, pepsin. Wagman ( 1 9 6 3 ) i s o l a t e d a t o x i c , d i a l y s a b l e subunit with a molecular weight o f 3,800 from a p e p t i c d i g e s t of type A t o x i n which was prev-i o u s l y t r e a t e d a t a pH of about 9. Bovey and Y a n a r i (i960) and H e r r i o t t (1962) have reviewed s t u d i e s on p e p s i n i n d e t a i l . I n essence, p e p s i n i s an endopeptidase which c a t -a l y s e s the h y d r o l y s i s of a wide v a r i e t y of peptide bonds but those between adjacent aromatic amino a c i d r e s i d u e s are e s p e c i a l l y s e n s i t i v e . The presence o f an amino group adjacent to the s e n s i t i v e bond i n h i b i t s enzymatic h y d r o l -y s i s . The optimum pH i s near 2.0 but a t t h i s a c i d e n v i r -onment i t i s subjected to a u t o l y s i s . Maximal s t a b i l i t y i s a t pH 5'0-5.5* and the enzyme i s r a p i d l y denatured a t pH o f about 6.0. Dolman ( 1 9 6 4 ) r e p o r t e d that type E t o x i n when d i g e s t e d by pepsin a t pH 2.5 r a p i d l y d e c l i n e d i n t o x i c i t y ; whereas a t pH 5*5 there i s no d e t e c t a b l e e f f e c t on i t s potency except under prolonged d i g e s t i o n . The s i g n i f i c a n c e of these r e s u l t s i s v e r y d i f f i c u l t to assess 12 as the same author made no e f f o r t to i s o l a t e and compare the toxic fragment a f t e r peptic digestion at these pHs. C. Tryptic digestion In the intestine, the toxin molecule i s subjected to further digestion by other endopeptidases as well as exopeptidases. The extent of digestion by the l a t t e r i s d i f f i c u l t to speculate on, but s i g n i f i c a n t digestion by endopeptidases p r i o r to i n t e s t i n a l absorption i s highly probable. The s p e c i f i c i t y of trypsin's attack on peptide bonds to which an arginine or l y s i n e residue has contrib-uted to the carboxyl group has been well established with synthetic and other highly p u r i f i e d protein substrates (Neurath and Schwert, 1950). The optimal pH range for trypsin a c t i v i t y i s 7-9 (Green and Neurath, 1954) but the enzyme also r e a d i l y undergoes autolysis at t h i s pH range. It has maximal s t a b i l i t y at pH 2.3 at 30 C. (Neurath and Schwert, 1950). Type E toxin i s not r e a d i l y inactivated by trypsin but i t s t o x i c i t y may be activated or potenti-ated (Duff et a l . , 1956). This phenomenon has been both speculated on and investigated recently by two independ-ent groups. The Japanese workers, Sakaguchi et a l . , (1959, 196l) have speculated about a toxin "precursor" molecule whose toxic moiety was masked by a group assoc-iated with nucleic a c i d . They believed that t h i s masking or i n h i b i t i n g group i s cleaved from the toxic moiety i n t r y p t i c digestion and the "activated" toxin molecule i s 13 i e released. They further claimed that both the "precursor" and the "activated" toxin have molecular weights of approx-imately 200,000 (Sakaguchi et a l . , 1964). The Canadian investigators, G-erwing et a l . , (1961, 1962) showed that t h e i r p a r t i a l l y p u r i f i e d type E toxin, both before and a f t e r trypsin treatment, was devoid of nucleic acid, and also pos-tulated that the process of t r y p t i c a c t i v a t i o n involves some degree of molecular fragmentation res u l t i n g i n the formation of a smaller, more active molecule. D. Chymotryptic digestion In the intestine, the toxin molecule must encounter the other endopeptidase, chymotrypsin. Even though t r y p t i c aotivation i s an i n v i t r o phenomenon, i t has been established that highly p u r i f i e d preparations of trypsin are contaminated with a trace amount of chymotrypsin (inagami and Sturtevant, I960). Chymotrypsin acts on a variety of peptide and ester linkages. The s p e c i f i c i t y of action has been discussed by Desnuelle (i960). The enzyme p r e f e r e n t i a l l y attacks peptide bonds which involve tyrosine and phenylalanine. I t has been reported that trypsin and chymotrypsin cause extensive degra-dation i n type A toxin (Coleman, 1954; H a l l i w e l l , 1954; Meyer and Lamanna, 1959); however no information i s available on chymotryptic treatment of type E toxin. MATERIALS AND METHODS Culture C l . botulinum type E, s t r a i n Iwanai was used through-out this study. This non-proteolytic, gas producing s t r a i n was found to be consistently toxigenic. Toxin production The type of medium, growth conditions, p u r i f i c a t i o n procedure and potency assay as described by Gerwing et a l . , (1961, 1964) were used. An average batch of toxin so ob-tained contained 30-50 mg protein with potencies ranging from 5,000-10,000 MLD per ml. The t o t a l volume was i n the region of 100 ml. Whenever subsequent procedures required changes i n pH and s a l t concentration of the toxin prep-arations, the l a t t e r were dialysed against the appropriate buffers or d i s t i l l e d water. In the same context, the toxin preparations were concentrated by the addition of saturated ammonium sulphate solution to a f i n a l volume of 60$ and allowed to stand at 4 C for 4-6 hours. The precipitate thus formed was collected by centrifugation at 5»000 X g and resuspended i n the desired amount of 0.01M acetate buf-f e r pH 4.5. Treatment of d i a l y s i s tubing Before use, a l l d i a l y s i s tubings (Visking Company) were boiled f o r 5 minutes i n a 0.01M solution of ethyl-enediaminetetraacetic acid (Versene) adjusted to pH 7.0, and then washed i n d i s t i l l e d water, except where otherwise 15 stated. Quantitative assay The toxin was assayed with the Folin-Ciocalteu phenol reagent based on the modification of Lowry et a l . , ( l 95 l ) • For comparison, the absorbance at 280 mu of 3.0 ml of 1:4, 1:3, 1:2 diluted and undiluted samples of a single "desalted" toxin preparation were measured with a Beckman model DU spectrophotometer. The toxin samples were then freeze-dried and t h e i r dry weights determined. The o p t i c a l densities were correlated with the dry weights. Potency assay Randomly bred Swiss white mice weighing approximately 20 gm + 5 were injected intraperitoneally with 0.05, 0.1, 0.2 and 0.3 ml of s e r i a l decimal d i l u t i o n s of the toxic samples. Dilutions were made i n s t e r i l e physiological s a l i n e . A single mouse per d i l u t i o n was used whenever an indicat i o n of the potency of the toxic sample was required. For accur-ate potency assays, groups of 5 mice per d i l u t i o n were used. The MLD was taken as the highest d i l u t i o n by which death occurred within 48 hours. Acid hydrolysis procedure Toxin samples of known concentration (0.2-0.5 uM) were dissolved i n 6N HC1 i n ampoules which were then sealed under vacuum. Hydrolysis was carried out for either 8 or 18 hours 16 at 105 C, a c c o r d i n g to the requirements of the r e s p e c t i v e procedures. Samples to be used f o r amino a c i d a n a l y s i s were washed t h r i c e i n d i s t i l l e d water a l t e r n a t e l y with f l a s h e v aporation a t 40 C. Ferfo r m i c a c i d o x i d a t i o n Performic a c i d was prepared by adding 1.0 ml of 30$ HgOg to 9.0 ml of 88$ formic a c i d . The mixture wa6 a l -lowed to stand a t room temperature f o r 1.0 hour and was then cooled to 0 C. D r i e d t o x i n samples of known concen-t r a t i o n to be analyzed f o r c y s t e i n e and methionine r e s i d u e content were o x i d i z e d with 2.0 ml of performic a c i d a t 0 C f o r 18 hours. Excess performic a c i d was removed by the a d d i t i o n o f 0.3 ml of 48$ HBr and the mixture was subjected to f r e e z e - d r y i n g and h y d r o l y s i s as d e s c r i b e d above. Amino a c i d a n a l y s i s Q u a n t i t a t i v e amino a c i d analyses were c a r r i e d out on a Spinco automatic amino a c i d a n a l y s e r a c c o r d i n g to the methods as d e s c r i b e d by Spackman et a l . ( l 9 5 8 ) . N-terminal amino a c i d determinations The l a b e l l i n g o f the N-terminal amino a c i d r e s i d u e s with Sanger's reagent (PDWB), a c i d h y d r o l y s i s and paper chromatographic i d e n t i f i c a t i o n as d e s c r i b e d by F r a e n k e l -Conrat et a l . (1955) were employed. 17 Inactivation by iodoacetate The reaction was carried out at pH 2.5 and 5.5. In both oases, approximately one mg of toxin was used. For pH 2.5t the toxin was adjusted to the required pH with 0.01 N HC1; for pH 5.5 - the toxin was pre-dialysed against 0.01 M acetate buffer at pH 5-5. The toxin samples so treated were halved. Excess iodoacetate (obtained from the Department of Chemistry, U.B.C.) dissolved i n d i s t i l l e d water was added to one half; the same volume of d i s t i l l e d water was added to the other half f o r control. These were allowed to stand at 25 C and the b i o l o g i c a l a c t i v i t y of 0.1 ml aliquots were taken and assayed at timed i n t e r v a l s . Inactivation by p-ohloromercuribengoate (FCMB) The reaction of FCMB (Calbiochem) with toxin was followed spectrophotometrically at pH 7.0 by a procedure modified from that as described by Boyer (1954) using cysteine as the standard. FCMB was dissolved i n the minimal amount of 0.001 N NaOH and made up to a f i n a l volume of 10 ml with 0.1 M phosphate buffer pH 7.0 (l,000yM FCMB per ml). Aliquots were taken and diluted to graded Jf molar concentrations. The l a t t e r were added to toxin samples of constant, known amounts (15-20JM per ml). Toxin samples were taken from a single batch of pure toxin i n each run. The reaction was carried out at 25 C. The extinction at 250 mu of the reaction 18 mixtures was measured with a Beckman model DU spectro-photometer immediately a f t e r the additions and thorough mixing of FCMB; and subsequently at 1,2 and 3 hours. Blanks comprised of the respective graded excess of FCMB solution mixed with phosphate buffer i n the same volume as the toxin were used to zero the spectrophotometer pr i o r to each measurement of the reaction mixtures. Spectrophotometric readings of the reaotion mixtures were corrected by the toxin's absorption at 250 mu. Toxicity was assayed when the reaction mixtures showed no further change i n d i f f e r e n t i a l absorption. Controls were made up of toxin mixed with the phosphate buffer. Chromatographic techniques Sephadex dextran gel (Pharmacia Pine Chemicals, Uppsala, Sweden) of d i f f e r e n t types were used. The dextran gels were hydrated i n the appropriate buffers or d i s t i l l e d water overnight at 25 C. Pines were d i s -carded by repeated decantations. The evenly sedimented dextran gels were then packed into t h e i r respective columns: For the i s o l a t i o n of a DDPS-peptide, Sephadex G-25 (fine) dextran gel i n d i s t i l l e d water was packed into a column 100 X 1 cm. The gel packed to a height of 93 cm was washed with 100 ml of d i s t i l l e d water and se t t l e d to a f i n a l height of 80 cm. Plow rate was not regulated. For the fractionation of a trypsin-activated toxin, 19 Sephadex G—75 dextran gel i n 0.05 M acetate buffer pH 4.5 was f i r s t packed into a column 110 X 0.8 cm, the gel bed was washed with 200 ml of the same buffer and set t l e d to a height of 42 cm. S i m i l a r i l y prepared Seph-adex G—50 (fine) dextran gel was then supraimposed to a f i n a l height of 105 cm. The whole composite gel bed was washed with 500 ml of the same buffer. Flow rate was regulated to 7.5 ml per hour. For the analysis of chymotrypsin-trypsin fragmented toxin, Sephadex G-25 (fine) dextran gel i n 0.05 M acetate buffer pH 4.5 was packed into a column 180 X 0.8 cm. The gel bed was washed with 200 ml of the same buffer and s e t t l e d to a height of 160 cm. Flow rate was regulated to 15 ml per hour. Col l e c t i o n of a l l eluted material as 2.5 ml fractions was accomplished i n a model V-10 Fraction Collector (Oilson Medical Electronics, Middleton, Wis.). A l l eluted fractions were analysed f o r the presence of 280 mu absorbance. In the i s o l a t i o n of a DDPS-peptide, the absorbance at 440 mu was also analysed. In the i s o -l a t i o n of a trypsin-activated toxin, the presence of nin-hydrin positive material was analysed thus: Ninhydrin (5$ i n acetone) was added i n a 1:1 r a t i o to the samples to be tested (usually 1.0 ml of each). The tubes were heated f o r 15 minutes at 100 C then immediate-l y cooled. The reaction tubes were diluted 3:2 with 95$ ethanol and read at 570 mu with a Beckman B spectrophotometer. 20 In the i s o l a t i o n of a chymotrypsin-trypsin frag-mented toxin, the presence of ninhydrin positive material was analysed according to the method described by Morris (1961): 2 gm of ninhydrin was added to 10 ml d i s t i l l e d water and 6.0 ml 0.1 M acetate buffer pH 5.0 i n 170 ml 95$ ethanol. This ninhydrin solution was then made up to a f i n a l volume of 200 ml with 95$ ethanol, with 2.0 ml 0.1 M CdClg added p r i o r to use. 5*0 ml of the ninhydrin reagent was added to the samples (0.5 ml) to be tested. The reaction tubes were heated for 30 minutes at 100 C and then cooled. The extinction at 500 mu of each tube was then measured with a Spectronic 20 (Bausch & Lomb Inc., Rochester 2, New York). Absorbance at 225 mu by the chymotrypsin-trypsin fragmented toxin was also tested. Preparative paper chromatographic technique Whatman's #1 paper was pre-washed with Partridge's solvent, the upper phase of a mixture of n-butanol, g l a c i a l acetic acid and water (4:1:5» V/v/V ). Having dried the paper at 40 C overnight, the sample was spotted as a narrow band at the o r i g i n . The paper equilibrated for h a l f an hour was run with the same solvent as the descending mobile phase. 21 E l u t i o n from chromatographic paper The desired material, detected by i t s yellow colour, was cut out.of the paper chromatogram and wrapped i n a piece of t i n f o i l . S u f f i c i e n t amount of eluting agent, d i s t i l l e d water was allowed to impregnate the paper by ascending c a p i l l a r y action. The whole bundle was secured by means of a rubber band onto an acid cleaned broth tube and spun f o r 5 minutes at approximately 1,000 rpm i n a c l i n i c a l centrifuge. The eluted sample was collected from the bottom of the broth tube. Usually the procedure was repeated f o r complete elution. Isolation of a DDPS-peptide 20-25 mg of toxin were treated with 0.05 ml of mercaptoethanol and allowed to stand at 37 C overnight. The pH of the toxic sample was adjusted to 7.2 with 1.0$ NaHCO^ and an excess of N-(4-dimethylamino-3»5-dinitro-phenyl)maleimide or DDPM (obtained from Dr. G.H. Dixon, Department of Biochemistry, U.B.C.) which was dissolved i n methylcellosolve, added. The l a b e l l i n g was allowed to take place at 25 C, followed by exhaustive d i a l y s i s of the material against d i s t i l l e d water. The d i a l y s i s tubing was not pre-treated with versene* The DDPM-lab e l l e d toxin was then digested by 1.0$ trypsin by weight (2 X c r y s t a l l i s e d , N u t r i t i o n a l Biochemioals Corporation) overnight at pH 7.5 and 37 C. The t r y p t i c digest was then eluted with d i s t i l l e d water through a 22 Sephadex 0—25 (fine) dextran gel column. The retarded materials were pooled, re-trypsinized under the same conditions as before and again eluted with d i s t i l l e d water through the same Sephadex column. The most re-tarded, yellow coloured material was collected, concen-trated by f l a s h evaporation at 40 C, and separated chromatographically with pre-washed Whatman's #1 paper using Partridge's solvent as the descending mobile phase. The DDPS-peptide separated i t s e l f from the other ninhydrin positive peptides by running close to the solvent front. The amino acid content as well as the N-terminal amino acid residue of the yellow DDPS-peptide, eluted o f f the paper chromatogram with d i s t i l l e d water, were analysed as previously described. Trypsin a c t i v a t i o n Trypsin (2 X c r y s t a l l i s e d , N u t r i t i o n a l Biochemicals Corporation) dissolved i n 1 M ammonium acetate buffer pH 5.8 was added to the toxin i n an approximate micro-molar r a t i o of 1:120 (assuming the molecular weight of the toxin to be 18,000 and that of trypsin to be 24,000). The pH of the preparation was adjusted to the required l e v e l , which was either 5.8 or 7.5. The mixtures were incubated at 37 C for 24 hour period to determine when maximal act i v a t i o n occurred and to delineate the degrad-ation curve. At various time intervals (5,15and 30 min-utes; 1,2,3,4,5»6,9 and 24 hours), samples were removed 23 f o r t i t r a t i o n . They were ten f o l d d iluted i n 0.05 M acetate buffer pH 4.0 to i n h i b i t further t r y p t i c digestion. Chvmotrypsin-trypsin degradation Alpha chymotrypsin (3 X c r y s t a l l i s e d , N u t r i t i o n a l Bioohemicals Corporation) dissolved i n 1.0 M ammonium acetate buffer pH 5.8 was added to the toxin i n an approximate micromolar r a t i o of 1:60 (assuming the molecular weight of the toxin to be 18,000 and that of the chymotrypsin to be 25,000). The mixture was incubated overnight at 37 C, a f t e r which an aliquot of 0.1 ml was taken and i t s potency assayed. Likewise, chymotrypsin was again added and incubated f o r a further 12 hours. Trypsin was added thrice to the chymotryptic digest i n the manner as described i n trypsin activation, at 5 hour i n t e r v a l s . P r i o r to each addition, the potency of the digest was assayed. Isolation of the trypsin activated toxin 5 hour, trypsin-treated sample containing 3-5 mg toxic material i n 10 ml quantities were added to the composite Sephadex Gr-75 and G—50 column. The toxic material was eluted with 0.05 M acetate buffer pH 4.5. 24 I s o l a t i o n of the chymotrypsin-trypsin fragmented toxin 5 hours a f t e r the second addition of trypsin, the digest i n toto. containing 5.5 mg toxic material i n a volume of 20 ml was added to the Sephadex G-25 column. The same method of elution as above was used. The amino acid contents and the N-terminal amino acid residues of the i s o l a t e d trypsin activated toxin and the chymotrypsin-trypsin fragmented toxin were determined. RESULTS AND DISCUSSION Quantitative assay It was not possible to conduct the whole study with a single batch of toxin. The quantity of toxin produced varies from batch to batch. This variance d i r e c t l y affects the potency of the toxin. To standardize the quantity of toxin produced each time implies the standardization of one or more of the following: growth conditions, quality of the media, inoculation and harvesting procedures and contamination by other organisms. For quantitative com-parison and stoichiometric evaluation of any chemical reaction, i t i s necessary to know the concentrations of the reactants p a r t i c i p a t i n g i n the reaction. Hence a rapid and convenient means of quantity assay f o r the toxin has to be devised. The amount of a given protein i s usu-a l l y determined i n d i r e c t l y by the Micro-Kjeldahl method of t o t a l nitrogen, or by the Lowry method of t o t a l protein; the Biuret and ninhydrin reactions have also been used (Kabat and Meyer, 196l). Each method cited has li m i t a t i o n s and disadvantages. The t o t a l nitrogen determination method does not discriminate between nitrogen of the toxin and that originating from the ammonium sulphate used i n the toxin p u r i f i c a t i o n process. The ammonium sulphate could not be dialysed free completely. The methods f o r t o t a l protein determination involve comparison of colour devel-opment between the standard and the protein reacted with the respective reagents. The standard can be any suitable homogeneous protein preparation containing aromatic amino 26 acid residues, especially tyrosine and tryptophan; f o r example egg albumin i s one of the advocated standards as i t i s considered to be a representative protein. The method of protein estimation with the P o l i n -Ciocalteu phenol reagent i s the method of choice as i t has been found to be expedient f o r the estimation of soluble protein with an acouraoy range of 10 to 100 ug. The method i s based on the oolorimetric measurement of the blue colour produced by the addition of the phenol reagent to an alkaline solution of protein. The colour i n t e n s i t y produced by a given amount of protein i s c h i e f l y a function of i t s tyrosine and tryptophan content. How-ever, other factors are known to play a role, especially the length of time to which the protein i s exposed to the a l k a l i p r i o r to the addition of the phenol reagent, and the presence of -SH and other reducing groups. Shoa-Chia and Goldstein (i960) found that any peptide bond w i l l y i e l d some colour, but certain amino acid sequences, not necessarily containing aromatic residues, which are more chromogenic than others l a r g e l y contribute to the colour y i e l d of the protein. Unlike the method of t o t a l nitrogen determination, contaminating ammonium sulphate gives no interference. Proteins show cha r a c t e r i s t i c absorption at 270-290 mu with a maximum at about 280 mu. This absorption has been attributed c h i e f l y to the contents of aromatic amino acid residues, especially tyrosine and tryptophan (Smith, 1929). 27 FIGURE 1 Quantitative assay of toxin. Symbols: ____ Lowry determination at 500 mu Spectrophotometric determination at 280 mu 28 The toxin absorbs strongly at 280 mu. The correlation of the toxin's absorption at th i s u l t r a v i o l e t region with i t s dry weight may provide a comparison and check for i t s quantitative assay. In figure 1, the quantitative assay f o r the toxin with the two di f f e r e n t methods are compared and related to the o p t i c a l densities of d i f f e r e n t amounts of toxin at 280 mu. At any given o p t i c a l density at 280 mu f o r a part-i c u l a r sample of toxin, the Lowry method indicated 50$ less toxin than that by the spectrophotometric method. The high r e s u l t obtained i n the l a t t e r method i s probably due par-t i a l l y to the contaminating, undialyzable s a l t which i s s i g n i f i c a n t i n ug amounts, and also to the inaccuracies of the weighing procedure. On the other hand, the low r e s u l t obtained i n the Lowry method cannot be regarded as absol-utely accurate. The accuracy of t h i s method i s c h i e f l y a function of the resemblance between the standard, egg a l -bumin and the protein to be estimated, the toxin. Consid-ering the inaccuracies inherent i n both the methods employed and the use of the calculated molecular weight of the toxin f o r converting the toxin into gram molar quantities; i t was decided to use the results as obtained by the more r e l i a b l e Lowry method f o r subsequent work with the toxin. 29 Inactivation by iodoacetate In the absence of -SH groups, the confinement of al k y l a t i o n by iodoacetate of the reactive side chains of certain amino acid residues, by the change of pH, was found to be e f f e c t i v e . However, when -SH groups are pres-ent i n the protein, r e s t r i c t i o n of the a l k y l a t i o n reaction i s complicated by the concurrent a l k y l a t i o n of -SH groups at the dif f e r e n t pHs. When the toxin was reacted with iodoacetate, the t o x i c i t y decreased by approximately 50$ at both pH 2.5 and 5*5. Should -SH groups be absent, a l k y l a t i o n would be confined to the thioether S of the methionine residues at pH 2.5; and to the imidazole group of h i s t i d i n e at pH 5*5* I f the single h i s t i d i n e residue i s associated with t o x i c i t y , i e . i n the region of the active centre of the molecule, then i t s a l k y l a t i o n should y i e l d more than the observed 50$ in a c t i v a t i o n . On the other hand, i f the -SH groups of cysteine residues were alkylated but at a greatly reduced rate, as would be expected at these pHs, then the observed i n a c t i v a t i o n may be mainly due to the a l k y l a t i o n of -SH groups. T n ^ e t i v n t i 9 n by PCMB Repeated determination of the number of cysteine residues i n the toxin molecule by quantitative amino acid analysis of performic acid oxidized and subsequent aoid hydrolysed samples of the toxin, has revealed that cysteine 30 TABLE 1 Reaction of Toxin with PCMB at pH 7.0 Molar r a t i o of PCMB to toxin Moles mercaptide formation per mole of toxin Percent i n a c t i v a t i o n 3*1 5:1 10:1 20:1 0.48 0.68 0.80 0.88 50 50-75 75 80 31 occurs as a single residue. The chemical modification by PCMB of the -SH group i n the toxin molecule i s not only s p e c i f i c but the reaction may be followed quantitatively. Preliminary experiments have shown that an excess of FCMB was necessary f o r mercaptide formation between the reactants. This may be due to the masking of the -SH group by the unknown secondary and t e r t i a r y structures of the toxin. I f the intention was the quantitative assay of cysteine i n the toxin, then denaturation or p a r t i a l enzymatic dig-estion of the toxin p r i o r to the reaction with PCMB becomes a prerequisite step. However, the object was to determine the extent of i n a c t i v a t i o n of the toxin when i t s cysteine residue has been s p e c i f i c a l l y modified. Only 80$ i n a c t i v -ation was observed when the toxin was reacted with approx-imately 20 f o l d molar excess of PCMB. The mercaptide formation between the toxin and the FCMB was never 100$ when compared with that of the standard, cysteine and PCMB. P a r a l l e l r e s u l t s can be seen i n table 1 between the extent of mercaptide formation and the percent i n a c t i v a t i o n of the toxin. Inaccuracies are due to the errors inherent i n the procedures of the spectrophotometrie measurements, of the b i o l o g i c a l assay and of the i n i t i a l quantitative assay of the toxin. Considering the comparative inaccur-acies between the methods of spectrophotometric determ-ination and b i o l o g i c a l assay, the results showed remarkable concordance. The experiment demonstrated that the reactive -SH 32 group of the cysteine residue, when s p e c i f i c a l l y modified by the t h i o l reagent PCMB, resulted i n the loss of biolog-i c a l a c t i v i t y of the toxin. Possible interpretations of the results are that the -SH group i s essential and that the cysteine residue i s located i n the active centre of the molecule. Such interpretations imply that the -SH group i s intimately related to the mode of action of the toxin. However, no data confirming t h i s speculation are as yet available. Bearing i n mind that the cysteine was tagged with a r e l a t i v e l y large group and that the amino acid residues adjacent i n sequence need not necessarily be adjacent i n space, i t i s also possible that the cysteine residue may be located very near to the active centre of the toxin molecule rather than i n i t . 33 I s o l a t i o n of a. DDPS-peptide Having established a relationship between the cysteine residue with the b i o l o g i c a l a c t i v i t y of the toxin molecule, l a b e l l i n g of the cysteine residue should f a c i l i t a t e the i s o l a t i o n of a small peptide from that predetermined region of the toxin molecule. To ensure the l a b e l l i n g of a l l the toxin molecules with the -SH group s p e c i f i c , yellow colored reagent, DDPM, mercaptoethanol was added p r i o r to the l a b e l l i n g to reduce the cysteine residues. Excess DDPM and lab e l l e d molecules of mercaptoethanol were mostly eliminated by d i a l y s i s before subjecting the l a b e l l e d toxin to t r y p t i c digestion. Since the toxin has nine residues of lysine and three of arginine, as revealed by quantitative amino acid analysis (table 2), exhaustive t r y p t i c digestion t h e o r e t i c a l l y should give r i s e to 13 peptides. Because of the s p e c i f i c i t y and r e p r o d u c i b i l i t y of cleavage, trypsin i s the enzymatic agent of choice f o r the fragmentation of the labe l l e d toxin. Por the preliminary f r a c t i o n a t i o n of the t r y p t i c digest which was water soluble, gel f i l t r a t i o n through a Sephadex Or-25 (fine) column was chosen, employing d i s t i l l e d water as the eluent. Wheaton'and Bauman (1953) have described i n d e t a i l the weak ion exchange properties and the absorption effects which are pronounced i n t i g h t l y cross-linked gels such as G-25. The th e o r e t i c a l basis, p r a c t i c a l consider-ations and experimental technique of fractionation of pro-teins, peptides and amino acids by gel f i l t r a t i o n have been 34 4 35 FIGURE 2a Fractionation of the once-trypsinized DDPM-labelled toxin by Sephadex G-25 FIGURE 2b Fractionation of the twice-trypsinized DDPM-labelled toxin by Sephadex G-25 Symbols: Absorbance at 280 my Absorbance at 440 mu 0.7 0.G i t * 0.5 0.3 0.2 36 discussed by G-elotte ( 1 9 6 4 ) . The process of fractionation of the once trypsin-digested toxin by gel f i l t r a t i o n i s i l l u s t r a t e d i n figure 2 a . The retention of the eluted y e l -low material indicated that fragmentation of the la b e l l e d toxin had occurred. The two peaks indicated that not a l l the molecules had been cleaved to the same extent. Re-digestion by trypsin of the pooled, eluted yellow material should cleave l a b e l l e d fragments of various sizes into more uniform fragments of s i m i l a r s i z e . This i s evident i n f i g -ure 2 b , a t y p i c a l elution p r o f i l e of fractionation of the twice t r y p t i c digested l a b e l l e d fragments. The two figures, 2 a and 2 b , i l l u s t r a t e the e f f i c a c y of cleavage by trypsin and the fractionation of the digests by gel f i l t r a t i o n using d i s t i l l e d water as the eluent. Further fractionation of, the g e l f i l t e r e d , l a b e l l e d preparation was performed by descending paper chromatography. The DDPS-peptide ran very near to the descending solvent front away from at least four other ninhydrin positive pep-tides which migrated to a position on the paper nearer the o r i g i n rather than the solvent front. The DDPS-peptide so obtained was analysed i n terms of i t s amino acid content and N-terminal residue. At least two factors complicated the quantitative amino acid analysis of the peptide. When the toxin was l a b e l l e d with DDPM, the carbonyl group i n the adduct tends to condense with the amino group at alkaline pH to form a c y c l i c compound. The l a t t e r breaks down into at least two moieties during acid 3 7 hydrolysis. However, repeated analyses gave results which indicated that the peptide has the probable amino acid con-tent as shown i n table 2. The homogeneity of the peptide was indicated by the presence of only one N-terminal residue, alanine. The l a t t e r was found by examination of the 8- and 18-hour hydrolysates of the dinitrophenylated peptide sam-ple. Because the peptide i s a product of trypsin digestion, the C-terminal residue may be either arginine or l y s i n e . Quantitative amino acid analysis ruled out the former. Determination of the C-terminal residue by carboxypeptidase B which i s s p e c i f i c f o r the further degradation of products of t r y p t i c degradation did not give clear cut, conclusive results but merely indicated that lysine i s the probable C-terminal amino acid residue. The amino acid sequence or the primary structure of the isolated dodeca-peptide has not been established. 38 Tryptic a c t i v a t i o n Previous studies (Gerwing et a l . , 1961, 1962) suggested that t r y p t i c a c t i v a t i o n of type E toxin involved some degree of fragmentation of the toxin molecule, but the mechanism has not been established. In order to i s o l a t e the trypsin-activated toxic frag-ment, i t i s essential to determine the conditions for max-imal a c t i v a t i o n of the toxin by t r y p s i n i z a t i o n . The pH range fo r the a c t i v i t y of trypsin and the l a b i l i t y of the toxin at alkaline pHs set the parameters fo r the experiment. A pH of 7.5 was selected because i t i s i n the region of optimal a c t i v i t y f o r trypsin although considerable inact-i v a t i o n of the toxin occurs. On the other hand, at pH 5.8 the toxin i n a c t i v a t i o n i s minimised and the t r y p t i c a c t i v -ation, although retarded, reaches a higher maximum potency. Reproducible results were obtained when the toxin was treated with trypsin at these pHs> Pigure 3a i l l u s t r a t e s the degree of potentiation of t o x i c i t y and the subsequent degradation of the toxin under the i n v i t r o conditions of treatment by trypsin. At pH 7.5, a sample of toxin with a potency of 10,000 MLB per ml was activated i n approximately 15 minutes to a maximal t i t r e of 200,000 MLD per ml with a subsequent rapid decline i n t o x i c i t y . At pH 5.8, the act-i v a t i o n process required about 5 hours and the maximal t i t r e observed was 300,000 MLB per ml. The subsequent decline i n t o x i c i t y was gradual. Trypsin p r e f e r e n t i a l l y attacks peptide bonds to which 39 FIGURE 3a Effect of pH and time on the t r y p t i o digestion of type E toxin Symbols: Toxin control at pH 7.5 _______ Toxin and trypsin at pH 7.5 Toxin control at pH 5.3 Toxin and trypsin at pH 5.8 40 an arginine or lysine residue has contributed to the car-boxyl group. Depending on the amino acid sequence and the t e r t i a r y structure of the substrate, some of these bonds may become p a r t i a l l y or t o t a l l y resistant (Thompson, I960). Apart from t h i s , the process of enzymatic cleavage i s random i n nature. Not a l l the toxin molecules may be cleaved to the same extent simultaneously, and some may even escape cleavage by trypsin. At pH 7 • 5» the rapid potentiation i n t o x i c i t y of the toxin may be attributed to the near optimal a c t i v i t y of the trypsin which was s u f f i c i e n t i n 15 minutes to reduce enough of the toxin molecules into smaller, more toxic fragments giving a t i t r e of 200,000 MLD per ml. There-after, progressive digestion continued to cleave more of the l y s y l and arg i n y l bonds, some of which are more intim-ately involved with the active s i t e of the molecule. The i n s t a b i l i t y of the toxin at pH 7.5 may coincidentally contribute to the subsequent rapid decline i n t o x i c i t y . At pH 5.8, t r y p t i c a c t i v i t y i s retarded and cleavage of the toxin molecule was at f i r s t confined to the most re a d i l y cleaves s i t e s . S u f f i c i e n t number of toxin molecules are transformed into smaller, more toxic fragments only a f t e r a r e l a t i v e l y longer period of 5 hours. The more potent t i t r e of 300,000 MLD per ml indicates that probably more toxin molecules have been s i m i l a r i l y reduced i n molecular s i z e . The subsequent, gradual decline i n t o x i c i t y may be due to the progressive cleavage of some of the p a r t i a l l y 41 FIGURE Fractionation of the toxin by Sephadex 3b trypsin-activated G-75 and G-50 Symbols: Absorbance at 280 mu Ninhydrin reaction read at 570 - - - Mouse MLD per ml 42 resistant bonds. The l a t t e r may have become susceptible with the change i n t e r t i a r y structure. The fragmentation process a f t e r maximal ac t i v a t i o n becomes too complicated and involved f o r further interpretation. Toxin samples were activated f o r 5 hours at pH 5.8 p r i o r to t h e i r fractionation by a composite Sephadex column. Because the molecular size of the trypsin-activated fragment to be isolated i s unknown, a column of Sephadex G-50 dextran gel superimposed upon G—75 with a diameter to height r a t i o of 1:130 was used. Sephadex G-50 has an approximate exclusion l i m i t of 10,000 and Sephadex G-75 - 50,000. Should the activated fragment be too large f o r retention i n the G-50 portion of the column, then some measure of fractionation may be achieved by the G-75 portion. The successful fractionation of the t r y p t i c digest i s i l l u s t r a t e d by figure 3b (a representative e l u t i o n p r o f i l e ) . The activated toxin can be i s o l a t e d from f r a c -tions 10-15. Quantitative amino acid analyses of the toxin molecule and the activated toxin (table 2) indicated that the l a t t e r has at least 18 amino acid residues fewer than the i n t a c t toxin molecule (even though the content of tryptophan and methionine residues have not been deter-mined f o r e i t h e r ) . The presence of a single N-terminal amino acid residue, arginine was taken as i n d i r e c t evidence f o r the homogeneity of the i s o l a t e d peptide. The N-terminal residue determination was performed by the examination of 43 8- and 18- hour hydrolysates, as well as the aqueous phase of the dinitrophenylated peptide samples by the paper chromatographic systems as previously described (Fraenkel-Conrat et a l . , 1955). The presence of the N-terminal arg-inine residue was further confirmed by the Sakaguchi s t a i n reaction. Closer inspection of table 2 revealed that the trypsin-activated toxin has two l y s i n e and one arginine residues fewer than the i n t a c t toxin. Perhaps, at maximal ac t i v a t i o n the remaining 7 l y s y l and 2 a r g i n y l bonds d i s -played r e l a t i v e resistance to cleavage by trypsin at pH 5*8. Within the l i m i t a t i o n s of the experimental procedures employed, the r e s u l t s indicate that there i s a lack of i d e n t i t y between the N-terminal amino acid residues and a difference i n the amino acid contents, i n the i n t a c t and activated toxin molecules. These results strongly suggested that the process of t r y p t i c a c t i v a t i o n involves the frag-mentation of the intact toxin giving r i s e to a smaller, more active molecule. 44 Chymotrypsin-trypsin fragmentation The trypsin a c t i v a t i o n experiment demonstrated that under certain i n v i t r o conditions, the toxin molecule may be fragmented without the loss of t o x i c i t y . The cross contamination of trypsin and chymotrypsin preparation has been established (inagami and Sturtevant, I960). In other words, even highly p u r i f i e d preparations of.trypsin are contaminated by chymotrypsin, and vice versa. Moreover, under i n vivo conditions, the two pancreatic endopeptidases should work s y n e r g i s t i c a l l y . These considerations led to the formulation of the hypothesis that the toxin may be further fragmented by the two endopeptidases under con-t r o l l e d , i n v i t r o conditions. Chymotrypsin, l i k e trypsin, also acts optimally on protein substrates i n the pH range 7.0-9.0. Preliminary experiments indicated that prolonged exposure of the toxin to chymotrypsin at pH 5*8 and 37 C resulted i n no detect-able loss of t o x i c i t y . The degradation curve, figure 4a, showed that there was no decrease i n potency when the toxin was exposed to two additions of chymotrypsin over a period of 24 hours, at pH 5.8 and 37 C. Since chymotryptic a c t i v i t y i s retarded at pH 5.8, perhaps only very susceptible peptide bonds, which are not involved i n the active s i t e of the toxin molecule, are cleaved. I t has been previously established that trypsin act-i v a t i o n at pH 5.3 requires approximately 5 hours. Figure 4a showed that there was a potentiation of t o x i c i t y i n the 45 FIGURE 4a E f f e c t of chymotryptic-tryptic digestion on the t o x i c i t y of type E toxin at pH 5.8 Symbols: Chymotryptic digestion • . Tryptic digestion Toxin control at pH 5*8 # Addition of chymotrypsin * Addition of trypsin 46 chymotryptic digest 5 hours a f t e r the f i r s t addition of trypsin. Subsequent additions of trypsin f a i l e d to f u r -ther potentiate or decrease the t o x i c i t y . The absence of a subsequent decline i n t o x i c i t y as observed i n t r y p t i c activation, i s open to speculation. Possibly the chymo-t r y p t i c digest gave r i s e to a toxic polypeptide whose conformation (or t e r t i a r y structure) permits only limited fragmentation by trypsin. This postulation seems to be ph y s i o l o g i c a l l y sound. Following fractionation of the chymotryptic-tryptic digest, the presence of ninhydrin positive, 280 and 225 mu absorbing material i n each f r a c t i o n was analyzed. Real-i z i n g that the ninhydrin reaction used i n the t r y p t i c act-i v a t i o n experiment i s at best only semi-quantitative, the quantitative method as described by Morris (l96l) was ad-opted. Because chymotrypsin p r e f e r e n t i a l l y cleaves pep-tide bonds involving aromatic amino acid residues, detec-t i o n of the presence of 280 mu absorbing material as a quantitative index of the fragmented toxin becomes inade-quate. At shorter wavelengths i n the u l t r a v i o l e t region, notably between 190-240 mu, the absorbance exhibited by aromatic amino acid residues i s overlapped with that by h i s t i d i n e , methionine, cystine and cysteine, as well as the peptide bond i t s e l f (Edsall, 1963). I t was a r b i t r a r i l y conceived that analysis of the presence of 225 mu absorbing material may serve as a useful, additional detection method. The chymotrypsin-trypsin fragmented toxin, taken from 47 FIGURE 4b Fractionation of the chymotrypsin-trypsin fragmented toxin by Sephadex G-25 Symbols: ______ Absorbance at 225 mu Absorbance at 280 mu — Mouse MLD per ml ..... Ninhydrin reaction read at 500 mu 48 the f i r s t peak (fractions 12-18) as shown i n figure 4b, was further characterized. Closer examination of the elution p r o f i l e of the chymotryptic-tryptic digest ( f i g -ure 4b) revealed that other fractions which are more re-tarded, also exhibit b i o l o g i c a l a c t i v i t y . As previously mentioned, retardation or retention i n gel f i l t r a t i o n indicates moieties of smaller molecular s i z e . No attempt was made to further characterize these smaller toxic frag-ments because of t h e i r probable heterogeneous nature, as indicated by the e l u t i o n p r o f i l e . Again the presence of a single N-terminal amino acid residue, t e n t a t i v e l y i d e n t i f i e d as valine, was taken as i n d i r e c t evidence f o r the homogeneity of the chymotrypsin-trypsin fragmented toxin. Quantitative amino acid analysis revealed that there was a considerable reduction of amino acid residues as compared with the i n t a c t toxin and the trypsin-activated toxin (table 2). Even though the quan-t i t a t i v e amino acid analysis f o r the chymotrypsin-trypsin fragmented toxin was incomplete, the reduction i n molecular size was obvious* 49 TABLE 2 Type E, toxin, trypsin-activated toxin and chymotrypsin-trypsin fragmented toxin compared i n terms of amino acid contents, N-terminal residues and comparative t o x i c i t i e s Chymotrypsin-Trypsin- trypsin Intact activated fragmented DDPS-Amino acid toxin toxin toxin peptide Cysteic acid 1 1 1 1 Aspartic acid 16 15 6 1 Threonine 7 7 3 Serine 8 8 4 3 Glutamic acid 11 11 5 1 Proline 8 8 3 Glycine 12 8 6 3 Alanine 6 4 4 2 Valine 6 4 1 Isoleucine 10 6 4 Leucine 10 8 4 Tyrosine 4 4 1 Phenylalanine 5 5 2 Lysine 9 7 * 1 Histidine 1 1 * Arginine 3 2 * Tryptophan * * • Methionine # * * N-terminal gly arg v a l a l a Comparative toxic 30-100 3-10 non-t o x i c i t y f o l d f o l d toxic Molecular 14,000- 10,000- 4,000- 1,100 weight 16,000 12,000 6,000 * not determined GENERAL DISCUSSION & CONCLUSIONS The investigation can be divided into two parts. F i r s t , chemical modification studies were employed to relate b i o l o g i c a l a c t i v i t y with the various amino acid residues i n the toxin molecule. Linking the f i r s t part to the second was the i s o l a t i o n of a DDPS-peptide from the toxin. In the second part, fragmentation of the toxin by proteoly t i c enzymes was attempted i n order to iso l a t e a toxic peptide of the smallest possible molecular s i z e . The random nature of the process of chemical modi-f i c a t i o n i s i l l u s t r a t e d by a l k y l a t i o n with iodoacetate. Even i f the a l k y l a t i o n reaction was confined to the re-active side chain of a pa r t i c u l a r amino acid residue by the manipulation of pH, concurrent a l k y l a t i o n of -SH groups at di f f e r e n t pHs made i t very d i f f i c u l t to assess the extent of modification of the toxin molecules, and to relate the l a t t e r to the loss of b i o l o g i c a l a c t i v i t y . The data obtained merely indicated that the single cys-teine residue may be associated with t o x i c i t y . On the other hand, modification of the cysteine residue with the -SH s p e c i f i c reagent, PCMB may be followed quantit-a t i v e l y . The amount of mercaptide formation which corresponded to the percent i n a c t i v a t i o n of the toxin indicated that the cysteine residue must be located at the active s i t e of the toxin, or very near i t . Schantz and Spero (1957) reported that PCMB inactivated type A toxin by 30$; but they f a i l e d to relate the b i o l o g i c a l 51 a c t i v i t y of the toxin to the cysteine residue. Studies i n progress (unpublished data) have revealed that FCMB inactivated the highly p u r i f i e d type A toxin obtained by Gerwing et a l . , (1965) by at l e a s t 90$ at pH 7.0. The same toxin has been shown to be devoid of tryptophan. The l a t t e r contradicts the erroneous claim by Boroff's group that the tryptophan residues are related to the t o x i c i t y of type A toxin (Boroff and DasGupta, 1965). It i s believed that there i s only one configuration fo r the active s i t e of each type of b i o l o g i c a l l y active molecules (Ram et a l . , 1962). The rest of the b i o l o g i c -a l l y active molecule (contributing structure), not i n -cluded i n the active s i t e may d i f f e r among the various types and species. Studies i n progress (unpublished data) have demonstrated that both types A and B toxins contain a peptide with the same amino acid composition as the DDPS-peptide of type E toxin. I f the i s o l a t e d DDPS-peptides form the active s i t e s of botulinus toxins, then t h e i r amino acid sequences should be more or less i d e n t i c a l . Moreover, maximal cleavage of the contributing structures of botulinus toxins should y i e l d a toxic pep-tide which must include those amino acid residues as i n the DDPS-peptide. The v a l i d i t y of the claim that the isolated dodecapeptide forms the active s i t e of botulinus toxins must await confirmation. The s i m p l i c i t y and s p e c i f i c i t y of the procedure f o r i s o l a t i n g the DDPS-peptide from type E toxin compared favorably with that 52 employed by Segal and Gold (1964) who fragmented t h e i r DDPM-labelled preparation (rabbit glyceraldehyde-3-phosphate dehydrogenase) with pepsin, and isolated a hexapeptide by means of electrophoresis at d i f f e r e n t pHs coupled with descending paper chromatography. The molecular weight of type E toxin, calculated on the basis of physical measurements, was reported to be 18,600 (Gerwing et a l . , 1964), whereas calculated from the number of amino acid residues, the molecular weight appeared to be between 14,000-16,000. Considering the errors inherent i n physical measurements and i n quantitative amino acid analysis, these calculated mol-ecular weights for type B toxin can be regarded as i n good agreement. Thus any fragmentation or cleavage of the contributing structure of the toxin molecule, must y i e l d a fragment with a reduced molecular weight. According to Sakaguchi et al.,(l964), both the toxin "precursor" and the trypsin-activated toxin have molecular weights of approximately 200,000. The results (table 2) c l e a r l y disputed the Sakaguchis' claim and further demon-strated that the process of t r y p t i c a c t i v a t i o n probably involves a removal of at least 18 amino acid residues from the N-terminus of the toxin molecule. The activated toxin has a molecular weight of 10,000-12,000, as c a l -culated on the basis of the number of amino acid residues. Besides demonstrating that cleaving away part of the con-t r i b u t i n g structure of the toxin molecule resulted i n a 53 change i n b i o l o g i c a l a c t i v i t y , the t r y p t i c a c t i v a t i o n experiment also showed that the toxin was not inact-ivated by l i m i t e d enzymatic hydrolysis. Incidentally, the b i o l o g i c a l a c t i v i t y of the trypsin-activated toxin became comparable to that of type A toxin which has a molecular weight of 1 2 , 2 0 0 . With a combination of chymotrypsin and trypsin, i t was possible to obtain a fragment of lesser molecular size ( 4 , 0 0 0 - 6 , 0 0 0 i n molecular weight) with maintenance of b i o l o g i c a l a c t i v i t y . 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