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UBC Theses and Dissertations

Studies on the active region of botulinus toxins : involvement and sequence of a peptide in the region… Van Alstyne, Diane 1966

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STUDIES ON THE ACTIVE REGION OF BOTULINUS TOXINSJ INVOLVEMENT AND SEQUENCE OF A PEPTIDE IN THE REGION OF THE SINGLE CYSTEINE RESIDUE IN BOTULINUS TOXINS TYPES A, B AND E by DIANE VAN ALSTYNE B.Sc. U n i v e r s i t y of B r i t i s h Columbia 1964 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Microbiology We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1966 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r -m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i -c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ^Tfld "C^^c^-Uo-^^ The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date / Mb ABSTRACT After establishing that» l i k e types B and~E toxins* type A botulinus toxin contains only one residue of cysteine per molecule* chemical modification studies were carried out showing that sulfhydryl-specific reagents l i k e p-chloromercuribenzoate (PCMB) and N-(4-dimethyl-amino-3»5-dinitrophenyl) maleimide (DDPM) cause a marked decrease in to x i c i t y . Types A> B and E toxins were reacted with DDPM and a labelled peptide was obtained from the tryptic digest of each of the toxins. The quantitative amino acid analyses of these three peptides were remarkably similar. Sequence analyses showed that a sequence ala-glu-ser-cys-ser-asp-ser i s common to a l l three DDPS-peptides. Type B botulinus toxin does not enzymatically alt e r acetyl-choline i n order to cause f l a c c i d paralysis. It i s postulated that the three thiol-containing peptides isolated contain a l l or part of a postulated active si t e common to each of types A, B and E botulinus toxins. TABLE OF CONTENTS Introduction , . i Review of the literature: I. Chemical modification of proteins 1 II. Concept of the active site 4 III. Determination of protein primary structure 6 IV. The role of sulfur in proteins; 9 Methodsi results and brief discussion: I. Culture and amino acid analyses 11 II. Purification and characterization of type A toxin 11 Results .13 Discussion 12 III. Chemical modifications 16 1. Reaction with iodoacetate (including results) 16 2. Reaction with PCMB (including results).... 17 3. Reaction with DDPM (including results) 18 Discussion of 1-3 19 IV. Isolation and amino acid analyses of DDPS-peptides 22 Results 24 Discussion 23 V, Sequence studies 27 Results 31 Discussion ...36 VI. Inactivation with acetylcholine 39 Results 39 Discussion 39 General discussion ...42 Conclusions • • 46 Appendix • .' 48 Bibliography FIGURE I: LIST OF FIGURES Amino acid analysis of typB A toxin page 13 FIGURE I I : Percent i n a c t i v a t i o n of C. botulinum types A and E toxins a f t e r reaction with iodoacetate 16 FIGURE I I I : Reaction of C. botulinum types A» B and E toxins with PCMB at pH 7.0 17 FIGURE IV: Percent i n a c t i v a t i o n of C. botulinum types: A, B and E toxins a f t e r reaction with an excess of DDPM at pH 7.0 18 FIGURE V: Representative amino acid analyses of DDPS-peptides from types A» B and E toxins 24 FIGURE VI: Sequence studies using technique #1 28 ACKNOWLEDGEMENTS This thesis could not have been completed without the advice and i n f i n i t e patience of Dr. G.H. Dixon and Dr. J . Black (Department of Biochemistry> U.B.C.) as w e l l as the t e c h n i c a l assistance BO generously given by Dr. J.H. Tremaine (Federal Department of Ag r i c u l t u r e Research S t a t i o n , Vancouver). I t i s impossible to f u l l y express the gratitude f e l t f o r the able supervision of Dr. J . Gerwing who, when confronted with any problem, was always w i l l i n g to give her undivided att e n t i o n and who i n v a r i a b l y a r r i v e d at the most sensible s o l u t i o n . INTRODUCTION The elucidation of the active sites of biologically active proteins is s t i l l a relatively new field. Few proteins are completely sequenced to date> certainly few of the large ones. The understanding of the interactions responsible for tertiary structure as well as quaternary structure* which is equally as important* is not complete as yet. Also* the search is only just beginning for chemical modifications of specific amino acids in order to look for resulting losses in activity. In view of these deficiencies i t is easy to understand why much of the work in the field of "active sites" relies on results which merely imply possibilities rather than point definitely to one answer. Disturbing the secondary* tertiary or quaternary structure of any protein during any investigation of the molecule's activity automat-ically imposes severe criticism of the interpretation of the results obtained. Yet i t is virtually impossible to state with certainty that any technique, regardless of how gentle* has not merely disturbed the configuration of the protein to result in loss of activity. Thus* a specific chemical treatment which affords loss in protein activity can-not alone be taken as evidence that the site modified is necessarily the active one in the molecule. It is important then* that more than one piece of data supports the hypothesis. Purified botulinus toxins are immunologically quite distinct, implying vast differences in t h e i r antigenic determinant groups. Also, their Blectrophoretic mobilities and amino acid analyses differ consid-erably. Yet, they are similar pharmacologically, indicating that a l l i i three toxins may have one region in common, namely the region around the active site. Only cysteine is present as a single residue in each of the three toxins, while histidine is present as a single residue in types B and E and as two residues in type A toxin. Interestingly both cysteine and histidine have been implicated in the literature as components of active regions of other biologically active proteins.. Because the toxin is stab-ilized by mercaptoethanol and pretreatment of dialysis sacs with versene prevents inactivation during dialysis, chemical modification of cysteine was investigated first. It was reasoned that i f the sequence of the residues around the single cysteine was common in a l l three toxins, then this would provide additional support'for the contention that these residues represent either part or all of the active site of the immunologically distinct botulinus toxins. . . 1 Review of the literature I. Chemical modification of proteins: Amino acid side chains may be modified to either detect result-ing losses in biological activity or to specifically label the chain so as to be able to isolate a peptide containing that label after fragmentation. There are few highly specific reegents for this purpose at present. Modification of SH groups: The sulfhydryl group of cysteine is highly reactive but only those reactions commonly used in inactivation or labelling studies will be reviewed. They can be divided into two types, substitution and addition reactions. The former type involves the use of organic mercurials (notably p-chloromercuribenzoate, or, PCMB) and those containing active halogens (commonly, iodoacetate and chloroacetate and their amides). The usefulness of PCMB (Hellerman, 1937) was enhanced in 1954 when Boyer developed a spec-trophotometric means of measuring the amount of mercaptide formed. Shantz and Spero (1957) used PCMB in inactivation studies on botulinus toxin and observed a loss of only 33% of biological activity which they discounted as insignificant. They concluded that thiols are not implicated in the toxic site. The salts of heavy metals like silver and mercury can be used for free thiol estimations as well as for inactivation. Many of the so-called "SH" enzymes like papain are inactivated by these salts and reactivated by thiols. The active halogen-containing reagents have been very useful in chemical modification studies, although they are not as specific as the org-anic mercurials. In the absence of cysteine alkylation can be confined to histidine at pH 5-6 and methionine at pH 2-3. Lysine, cysteine and methionine 2 are alkylated at pH 8-10 (Gundlach et al.» 1959). Inactivation of ribonuc-lease by iodoacetate was first attributed to alkylation of thiols (Zittle, 1946) but Hirs et al. (1958) showed that no cysteine was present in the enzyme, prompting Gundlach*s studies;. The addition reactions of SH groups commonly used for inactivation or labelling studies are those of maleimide derivatives. Maleic acid itself undergoes conversion to unreactive fumaric acid. Friedman et al. (1949) first used maleimide and N-ethylmaleimide (NEM). NEM has since been used extensively on such proteins as ficin (Liener, 1961), ovalbumin (Alexander, 1958), potato amylase (Speck et al., 1961) and hemoglobin (Benesch et al., 1961). The use of coloured maleimides has proven even more valuable. N-(2,4-dinitroanilino)maleimide was first used by Clark-Walker and Robinson (1961) while l\l-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) was reported by Witter and Tuppy (I960), both groups using these derivatives for studies on bovine serum albumin. DDPM has since been used to label essential thiols in glyceraldehyde-3-phosphate dehydrogenase (Segal and Gold, 1964) and various lactate dehydrogenases (Fondy et al., 1965) among others. Radioactively-labelly maleimide derivatives have also been used (Holbrook et al., 1965). Modicication of other amino acids: Photooxidation in the presence of methylene blue (Weil et al., 1957) will modify tryptophan, histidine, tyrosine, methionine, and cysteine residues although the oxidation may be partially pH-controlled. Although very non-specific, this technique has been useful in the studies on chymo-trypsin which contains two methionines and two histidine residues (Ray and Koshland, 1960; Schachter and Dixon, 1962 and 1964). Botulinus toxins 3 have been shown to be inactivated by photooxidation (Weil et al., 1957). Boroff and DasGupta (1965) used the pH-dependence of the reaction to relate toxicity to methionine, cysteine or tryptophan residues. Methionine may be specifically oxidized in chymotrypsin using H^ O^  at pH 3 (Koshland et al., 1962; Schachter and Dixon, 1964). After photooxidation experiments, Boroff and DasGupta (1964) used peroxide oxid-ation of botulinus toxin to conclude that methionine was not implicated in the toxic site. Koshland et al. (1964) have reported the reagent 2-hydroxy-5-nitrobenzyl bromide (HNBB) which is specific for tryptophan and cysteine. Boroff et al. (1965) used HNBB and attributed the resulting loss in activity to modication of tryptophan. They disregarded the possibility of cysteine modification leading to inactivation on the basis of the PCMB experiments, of Schantz and Spero (1957). A chemical modification of histidine and tyrosine residues (and other groups as well) has been reported (Horinishi et al., 1966) using diazonium-l-H-tetrazole (DHT). But no reagent specific for histidine has been reported to date. Another approach to specific modification of proteins involves the reaction with a substrate or substrate analogue. Lai et al. (1965) have used radioactively-labelled dihydroxyacetone phosphate to label fructose-1,6-diphosphate aldolase to label the active site. Reduction of the enzyme-substrate complex yielded a stable jS-glycerophosphate-protein derivative. These authors established the amino acid sequence around a labelled peptide obtained from tryptic digestion. 4 II. The concept of the active site; The results of inactivation studies may be misleading and perhaps too often the amino acid modified and its adjacent residues have been loos-ly termed the "active site" without the support of other data, and indeed, without ascertaining whether or not inactivation was due merely to distor-tion of tertiary structure. Fondy and his group (1965) inactivated their lactate dehydrogen-ases with p-hydroxymercuribenzoate (HMB). They then found that their in-activated enzymes were s t i l l able to cross-react immunologically 100% with the native forms andalso retained the same sedimentation coefficients, indicating that the inactivation was not due to dissociation into subunits. It would appear that only a limited number of amino acid side chains are intimately involved in the chemical events concerned in the biological activity of a given protein. But, large portions of the protein may be concerned with binding of the active sites to the receptors, or may possess functional significance not as yet defined. For example, both coenzyme-binding and substrate-binding "sites" (peptides) have been isolated from some of the dehydrogenases. Holbrook and Pfleiderer (1965) inhibited enzymatic activity of pig H^lactate dehy-35 drogenases by specifically labelling SH groups with N-(N-acetyl-4- S-sulfanoylphenyl)maleimide. This inhibition was completely prevented by the presence of reduced coenzyme but not by the presence of substrate. They isolated a thiol-peptide identical to that of Fondy et al. (1965). Di Sabato and Kaplan (1963), working on similar inactivation studies, reported identical findings. But, these authors point out that coenzyme may protect against labelling by a general stabilizing effect on tertiary structure 5 (Di Sabato and Kaplan, 1964). On the other hand, Harris et al. (1963) have isolated a peptide from glyceraldehyde-3-phosphate dehydrogenase that is thought to represent the substrate-binding site. Reaction of the en-zyme with any of the SH reagents results in loss of bound NAD and inactiv-ation (Segal and Boyer, 1953). This can be prevented by prior reaction of the enzyme with the substrate. Thiol esters have been postulated as intermediates in a number of enzyme reactions, implicating thiols as components of these active sites. These enzymes include glyceraldehyde-3-phosphate dehydrogenase (Racker, 1954), papain (Durell and Fruton, 1954), glyoxalase and and SH enzyme associated with acetylcoenzyme A and fatty acid synthesis (Cecil, 1963). The import-ance of thiols in other enzymes' functions has. not been as clearly demon-strated. The most definitive work has been done on the peptide hormones. The adrenocorticotropic hormone (ACTH) is known to have both adrenocorti-cotropic and melanocyte-expanding activities, while the melanocyte-expand-ing hormones (o^ -MSH and f$ -MSH) both contain a common heptapeptide sequence which is also found near the N-terminal of ACTH as well as in a lipolytic peptide (Astwood et al., 1961) known to have marked MSH activity. The in vitro synthesis of parts of this sequence (see appendix) has yielded active peptides. In this way i t has been possible to narrow down the components of the active site. Also studied have been the effect on act-ivity of expanding the chain length, altering side chains and changing stereochemistry of amino acid components in the chain (Hofmann and Katsoyann 1963). 6 III. Determination of protein primary structure: Logically, the first step must involve the destruction of second-ary and tertiary structures to produce a random coil in which all peptide bonds are equally exposed to cleavage. Through the use of many specific cleaving agents one may be able to deduce the region from which each frag-ment was derived and reassemble the smaller fragments in their proper order. Alternatively or simultaneously one may employ step-wise degredation tech-niques from the amino or carboxy terminals. Specific cleavage may be effected either enzymatically or chem-ically. Only the most commonly used methods will be reviewed. A. Trypsin is the only enzyme considered to be truly selective, cleaving only the peptide bonds whose carboxyl groups are contributed by either arginine or lysine. Boyer et al. (1960) have extensively reviewed the important features of trypsin's action. Adjacent arginyl or lysyl and adjacent negatively charged residues and the peptide bond between lysine and proline often prove resistant to the action of trypsin. However, the usefulness of tryptic cleavage can be enhanced by alkylation of cysteine residues with bromoethylamine to form S-(j3 -aminoethyl) cysteyl residues which then closely ressemble lysine and become susceptible to hydrolysis by trypsin (Lindley, 1956). Conversely, i t is also possible to selectively block lysine residues in order to prevent cleavage of their peptide bonds. DNFB (2,4-dinitrofluorobenzene) has been used by Redfield and Anfinsen (1956) but deblocking of the stable N-alkyl derivative formed is not possible. However, NH2 groups are easily regenerated when using CS^ (Merigan et al., 1962), methyl acBtimidate (Ludwing, 1962) or with the e t h y l t h i o l ester of trifluoroacetic acid (Goldberger and Anfinsen, 1962). 7 B. o^-Chymotrypsin hydrolyzes peptide bonds adjacent to aromatic amino acids preferentially while leucyl, methionyl, asparaginyl, glutamyl, and occasionally histidyl peptide bonds are also hydrolyzed, although at a much reduced rate. E.O.P. Thompson (1960) provides a good review of the activity and general characteristics of chymotrypsin. C. Papain, pepsin and subtilisin all appear to have a poorly defined specificity. Pepsin appears to favour aromatic amino acids' peptide bonds but many other amino acids are also susceptible. Papain and subtilisin are very non-specific and can reduce almost any protein to many small pep-tides. Sinn et al. (1956), using subtilisin to cleave glucagon, found that bonds involving glycine, threonine, aspartic acid, lysine, leucine, arginine and valine were hydrolyzed. All three enzymes are thoroughly reviewed by Thompson (1960) . D. Leucine aminopeptidase (LAP) can sequentially hydrolyze the pep-tide bonds of all the common L-amino acids in an N-terminal position except proline and cystine which are attacked very slowly (Spackman at al., 1955). Hill and Schmidt (1962) have shown that LAP used in conjunction with pro-lidase can sequentially hydrolyze peptides completely into their amino acids.. However, i t is most useful for the complete degredation of a given peptide for subsequent amide determinations as well as for the elucidation of se-quences of particularly labile amino acids like tryptophan (Canfield, 1963). Unfortunately, degredation can not be controlled, but, depends on the sus-ceptibility of the individual peptide bonds. E. Carboxypeptidases A and B hydrolyze the peptide bonds of carboxy-terminal amino acids. Carboxypeptidase A reacts very slowly on some pro-teins due to the steric hindrance attributable to the secondary and tertiary 8 structure of the substrate (Boyer et al., 1960), but is generally remarkably active (Halsey and Neurath, 1955; Nylander et al., 1959). Carboxypeptidase B facilitates the removal of carboxy-terminal lysine, argine or ornithine residues (Folk, 1956), as well as 5-(^ -aminoethyl)cysteine. Chemical reactions now available for the specific cleavage of proteins were reviewed by Witkop (1961). Such reactions include the use of l\l-bromosuccinimide, specific for tryptophyl and tyrosyl bonds (Patchornik et al., 1960; Schmir and Cohen, 1961); cyanogen bromide, specific for methionyl bonds; and dinitrofluorobenzene, specific for cysteinyl bonds (Patchornik et al., 1963). Hydrolysis of peptide bonds using strong mineral acids is an almost routine approach in sequence analysis, although the more acid-labile amino acids are lost and the amides are converted to the dicar-boxylic acids during hydrolysis. The sequential degradation using the Edman reaction (1950), in which the peptide N-terminal is identified as the phenylthiohydantoin deriv-ative, may be used in conjunction with the dansylation technique of Gray and Hartley (1963) employing thin-layer chromatography (Seilder et al., 1964) for amino acid identification (as diagrammed in figure VI). In this way the difficulties involved in the identification of the hydantoins (on paper cnf subtractively) are overcome and the technique is not only more rapid, i t also requires less material than the dinitrophenylation technique of Sanger (1945) 14 for example. However, the use of radioactive FDNB-C (Dreyer and Bynum, 1963) has enabled identification of an M-terminal amino acid with only 0.1-1.0 mumoles of sample. 9 IV. The.role of sulfur in proteins: Disulfide bridges may have a structural or a functional role. Lipoic dehydrogenase contains two SS bonds that are thought to be concerned in the electron transfer reaction (Massey and Veeger, 1961). Vasopressin reacts with tissues via a disulfide exchange reaction* perhaps altering the permeability of the cell membrane in some way (Schwarz et al., 1960). Sim-ilar results have been found studying insulin (Fong et al., 1962). A di-sulfide exchange reaction followed by a reduction with NADPH has been pos-tulated as a mode of action of hepatic glutathione reductase (Mize et al., (^ 1962). However, structural importance is considerable. The spontaneous return of the reduced molecule to its native configuration upon oxidation, or, the rearrangement of the chain to the conformation of lowest free energy is aided in part by the formation of disulfide linkages. Kendrew (1961) has shown that methionine side chains of myoglobin lie in the center of the molecule in association with other apolar groups, making i t passible that the role of methionine in proteins is a structural one. Vithayathil and Richards. (1961) have provided concrete evidence that methionine is instrumental in maintaining protein structure with their work on ribonuclease. And, the met-3 in chymotrypsin has been assigned a struc-tural role by Schachter and Dixon (1964). No functional role has yet been unequivocably established for methionine. Sulfhydryl groups are classed as either reactive or unreactive, and the latter are at present poorly understood. It is possible that ionization of the SH may govern reactivity for i t has been shown that sub-stitution reactions take place with the mercaptide ion rather than with the undissociated SH group (Rapkine, 1933; Dickens, 1933; C.V. Smythe, 10 1936'; Hagen, 1956). Ionization may in turn depend on the immediate envir-onment of the SH group. In a hydrophobic region of a protein, the free energy change of the ionization process will be increased and therefore the tendency to ionize decreased. The reactive SH groups in hemoglobin have been shown to be involved in labile intramolecular linkages (Gibson and Roughton, 1955) so that it's possible that a reaction with these groups could cause a change in config-uration, leading to a change in heme-heme interactions (Cecil, 1963). Many of the SH enzymes may undergo configurational changes when the thiol reacts in any way. For example, when carboxypeptidase loses its Zn+^, i t becomes inactive, although its physical properties have not changed, indicating a very small configurational change (Neurath, 1960). The change is probably confined to the active site since the inactive form does not bind the in-hibitor ^-phenylpropionate. The reaction of SH groups in those enzymes functioning via a thiol ester has been discussed. The role of the unreactive SH groups in proteins is s t i l l not clear. Although they appear to be covalently bonded in the undenatured protein (Linderstr^im-Lang et al., 1941), only comparatively mild treatments are sufficient to cause a reversal to normal reactivity along with denatur-ation, sugesting that they are taking part in apolar, or, hydrophobic bond-ing (Nemathy et al., 1962a, b) . Scheraga: estimated that in this respect the S should be equivalent to one CH^  group. Unreactive thiols are found within the two o^chains in horse hemoglobin (Cullis et al., 1962) and therefore within an apolar region. Cecil and Snow (1962 a, b) concluded that such thiols must be concerned in intrachain bonding. They are also important in maintaining the configuration of some enzymes (Cecil, 1963). 11 METHODS AND RESULTS Culture C. botulinum type A s t r a i n corn* type B s t r a i n Lamanna and type E s t r a i n Iwanai were used throughout. The medium, growth conditions, p u r i f i c a t i o n procedures and potency assay were as described by Gerwing et a l . (1961, 1966) and Van Alstyne et a l . (1966). Amino acid analyses Quantitative amino acid analyses were c a r r i e d out on a Beckman-Spinco automatic amino ac i d analyser according to the method of Spackman et al-. (1958). A l l samples to b B analysed i n t h i s way had been suspended i n 6N HC1, sealed under vacuum and hydrolysed f o r 24 hours at 105 C. A l t e r n a t i v e l y , a performic acid oxidation was performed p r i o r to hydroly-s i s . Performic acid was prepared by adding 1 ml of 30% ^2^2 ^° ^ °^ 88% formic acid and allowing the mixture to stand f o r 1 hour at 20 C before cooling to 0 C. Toxin samples of known concentration were o x i d -ized with 2 ml of performic acid at 0 C f o r 18 hours. 0.3 ml of 48% HBr was added to remove excess performic a c i d . The s o l u t i o n was then dried down and hydrolysis c a r r i e d out as before. P u r i f i c a t i o n and chara c t e r i s a t i o n of type A toxin P u r i f i c a t i o n was. c a r r i e d out according to the method of Gerwing et a l . (1965a) with the fo l l o w i n g two modifications: a/ Columns were packed to a f i n a l height of 25-30 cm. -b/ The p r e c i p i t a t e d toxin was resuspended i n citrate-phosphate buffer 12 at pH 5.6 rather than in c i t r i c acid. Quantitative amino acid analyses were carried out on both performic acid and HC1 hydrolysates as previously described. The results are shown in figure I. . Tryptophan was determined according to method K of Spies and Chambers (1949) and spectrophotometrically according to the method of Goodwin and Morton (1946). The analysis obtained agrees suprisingly well with that pub-lished by Buehler et a l . (1947) who arrived at a minimum molecular weight of 45,000 on the basis of their amino acid analysis. However, i f one combines their calculations for cysteine and -J-cystine and takes that ratio as 1, then a minimum molecular weight of about 15,000 could also be calculated for the crystalline toxin. These workers estimated cysteine by t i t r a t i n g the free sulfhydryls and estimated the -^cystine by reducing and then r e t i t r a t i n g the newly-formed sulfhydryls. It i s obvious that a certain amount of oxidation of their free -SH groups must have occured, perhaps during c r y s t a l l i z a t i o n , to afford them these results. Not only do the molecular weights of the crystalline toxin and the low molecular weight toxin of Gerwing et a l . approximate one another, there i s also a general s i m i l a r i t y in the ratios of the individual amino acids. • Repeated tryptophan determinations were done on 5 and 10 mg samples of toxin obtained from toxin solutions which had been dialyzed exhaustively against d i s t i l l e d water at 4 C and then freeze-dried. No trace of tryptophan was ever detected. Based on a molecular weight of 13 FIGURE I: Amino acid analysis of type A corn toxin Molar ratio (Minimal M.W.= Probable # amino acid about 15,000) of residues lysine 10.95 11 histidine . 2.00 2 arginine 3.95 4 aspartic acid 19.70 20 threonine 7.86 8 serine 7.79 8 glutamic acid 17.20 17 proline 4.64 5 glycine 14.25 14 alanine 13.95 14 valine 10.23 10 methionine 1.93 2 isoleucine 8.64 9 leucine 10.70 11 tyrosine 2.00 2 phenylalanine 5.50 6 cysteic acid 1.17 1 No trace of tryptophan was detected either with the Spies and Chambers or the Goodwin and Morton method. 1 4 1 5 , 0 0 0 » 5 and 1 0 mg samples contained 0 . 3 3 and 0 . 6 6 JJM respectively which according to Spies and Chambers, is more than adequate for determining tryptophan. The spectrophotometric analyses gave a negative value for^LIM tryptophan, close to zero, so that no residue number per toxin molecule could be calculated, indicating the absence of tryptophan. However, i t has been reported that when the tryptophan content of the molecular spec-ies under consideration is low, then inaccurate values may be obtained. (Light and Smith, 1 9 6 3 ) . However, the fact that absolutely no trace of tryptophan was ever detected using this method would indicate that i t is reasonable to assume that that amino acid is absent. These results are further substantiated by the fact that no tryptophan has been detected in type B Lamanna toxin after repeated analyses. The finding that type A toxin lacks tryptophan must be recon-ciled with the findings of Boroff and his group who have concluded that tryptophan is implicated in the active region of type A. toxin as prev-iously reviewed. It is felt that either the photo-oxidation caused deactivation of the toxin by disrupting tertiary structure or, more likely, by altering some residue other than tryptophan. It is possible that the sulfhydryl groups of cysteine were oxidized causing deactivation These workers also used 2-hydroxy-5-nitrobenzyl bromide (HNBB) to chemically modify type A. toxin. This reagent is specific for trypto-phan and cysteine. Unfortunately, the involvement of -SH groups was discounted on the basis of the PCMB experiments of Shantz and Spero 15 (1957) and therefore at t r ibuted loss of b i o l o g i c a l a c t i v i t y to s u b s t i t -ution in tryptophan. With the amino acid analyses demonstrating that , l i k e types E and B, type A toxin also contains a s ingle cysteine residue per toxin molecule, incentive was. given to continue the work already started on' the chemical modif ications with a view to future studies on the residues in the immediate v i c i n i t y of the s ingle modified cysteine in each tox in . 16 Chemical modifications: 1. Reaction with iodoacetate (IA): Types A and E toxins were reacted with a 100 fold molar excess of IA (obtained from the chemistry department, U.B.C.) at pH 5.5 and 2.5. At each pH one toxin sample was mixed with IA dissolved in water and an equal volume of distilled water was added to a second, identical toxin sample. Both samples were incubated at 20 C for 1 hour and 0.1 ml aliquots were removed and titrated in mice, using the appropriate controls. The results; are shown in figure II: FIGURE II: Percent inactivation of C. botulinum types A and E toxins after reaction with excess iodoacetate Percent Inactivation Toxin pH 2.5 pH 5.5 Type A 60 60 Type E 50 50 17 2. Reaction with p-chloromercuribenzoate (PCMB): A stock solution of PCMB (sodium salt, Calbiochem) was dissol-ved in a minimal amount of 0.001 IM NaOH and further dilutions were then made in M/15 citrate-phosphate buffer at pH 7.0. 5 ml of type E toxin (1 mg/ml) were mixed at room temperature with varying amounts of PCMB: molar equivalence, 2, 3, 5, 10 and 20 fold molar excesses. The rate of mercaptide formation was followed spectrophotometrically at 250 mp accord-ing to the method of Boyer (1954), using cysteine HC1 (N.B.Co.) as the standard. At each spectrophotometric reading, 0.1 ml of the reaction mixture was withdrawn and titrated using the appropriate controls. Sim-ilar studies on type A and B toxins were carried out at pH 7.0 using only a 10 fold molar excess, of PCMB. Results are shown in figure III: FIGURE III: Reaction of C. botulinum types A, Band E toxins with PCMB at pH 7.0 Molar ratio Moles mercaptide Percent Toxin PCMB/toxin formed/mole toxin. inactivation Type E 1:1 0.20 30 2:1 0.20 30 3:1 0.48 50 5:1 0.68 60 10:1 0.88 80 20:1 0.88 80 Type A, 10:1 0.95 90 Type B 10:1 0.92 99 IB 3. Reaction with N-(4-dimethylamino-3»5-dinitrophenyl) maleimide (DDPM)s All three toxins were treated with an excess: of mercapto-ethanol and then allowed to stand for 3 hours at room temperature. Lab-elling was carried outi after dialyzing out the excess mercaptoethanol, according to the method of Segal and Gold (1964!) at pH 7.0 for 2 hours at room temperature. The pH was adjusted with 0.1 N NaOH from 5.6 (used for the purification of types A and B) and 4.5 (used for the purif-ication of type E). A large excess of_DDPM was uded, dissolved in a min-imal amount of methyl cellosolve. After incubation, 0.1 ml aliquots were removed and assayed in mice, using the appropriate controls;. The results, are shown in figure IV: FIGURE IV: Percent inactivation of C. botulinum types A, B and E toxins after'reaction with an excess of DDPM at pH 7.0 Toxin Percent Inactivation Type A 90 Type B 99 Type E 90 19 Because alkylation by iodoacetate is not specifically confined to histidine at pH 5.5 or methionine at pH 2.5 when sulfhydryl groups are present in the protein under study, the results obtained with types A and E toxins indirectly point to the involvement of cysteine in the active site. Degrees of inactivation obtained using iodoacetate were high, but when compared to 80-90$ inactivation obtained with the highly specific PCMB and DDPM, they were not as high as one would expect i f either meth-ionine or histidine were the key residue in conferring toxicity on the whole molecule. But, one could interpret the results as. being due to the alkylation of the -SH group of cysteine at a reduced rate. This would seem to be the best interpretation in view of the other data ob-tained using chemical modifications and, indeed, in view of the DDPS-peptides obtained and sequenced. It was impossible to carry out the reaction at pH 8.5, optimal for reaction of PCMB with -SH groups (Goddard and Michaelis, 1935), as botulinus toxins are rapidly inactivated under even mildly alkaline conditions. Reaction of the toxins with PCMB afforded perhaps the best support for the contention that cysteine is somehow implicated in the activity of the toxin. As previously reviewed, PCMB is absolutely spec-i f i c for cysteine at pH 7.0, and the reaction can be followed spectro-photometrically. The correlation of loss of activity with mercaptide-formation strongly supports the hypothesis. 20; As previously reviewed, other workers (Shantz and Spero, 1957) used PCMB on their crystalline type A toxin and obtained 33% inactivation which they disregarded as; insignificant. However, i t is probable that few -SH groups were available for reaction after the aggregation and oxidation which accompanied their crystallization procedure. Two criticisms may be levelled at the chemical modification experiments. First, 100% inactivation was never attained and second, perhaps the loss in activity detected was due to some effect other than the labelling of the -SH groups. After reaction for one hour with a 10 fold excess of PCMB, mer-captide formation and deactivation cease to progress even though not a l l the -SH groups have been alkylated. It is possible that the remaining unreacted protein has aggregated to shield the contained cysteine res-idues so that no increase in PCMB concentration is able to drive the reaction to completion. It is interesting that each of the three toxins has; its own characteristic final degree of inactivation after identical reactions with the alkylating agent. The pH used is also of importance in connection with degrees of inactivation using either PCMB or DDPM. When each toxin was- reacted at the pH in which i t is eluted from the column at purification (type A at 5.6, type B at 5.6 and type E at 4.5) the loss of biological activity was not as great as that obtained at pH 7.0, when compared to controls. It appears that when the protein is near the pH i t meets in vivo, its sulfhydryls become more available for reaction. It is reasonable, then, that at the acid pH's of 4.5 and 5.6, these toxins should be most stable, 21 ie. the -SH groups are not as, available for reaction, possible due to a pH-dependent conformational change which has a shielding effect in an acid environment. The possibility that deactivation is merely due to an altera-tion in tertiary structure after labelling is a more difficult criticism to discount soley on the basis of the results, obtained to this point. It is also possible that the alkylated group has not disrupted tertiary structure but has merely interferred sterically with some other residue adjacent in space rather than in sequence, to cause deactivation. A final possibility, in the same vein, is that the labelled cysteine is close to, but not actually involved in, the active site. All of these criticisms have in common the arguement that the deactivation caused by labelling is due to some secondary effect, ie., due to some effect other than the actual labelling. As previously pointed out, only a second set of data from quite a different source can confirm cysteine's involvement in the toxic site. This data was provided by the following work with the DDPS-peptides obtained from tryptic digests; of the three labelled toxins. 22 Isolation and amino acid analysis of a DDP5-peptide from a l l three toxins The single cysteine residue in each toxin molecule was. reacted at pH 7.0 with DDPM, dialyzed exhaustively against d i s t i l l e d water, . t rypsin ized for 24 hours; at 37 C (using a 1% concentration of trypsin) at pH 7.5 , passed through a 100X0.9 cm Sephadex G-25 column, eluted with d i s t i l l e d water, and 3 ml f ract ions were col lected on a Gilson model V-10 f ract ion c o l l e c t o r . The s ingle orange f rac t ion so obtained was; retrypsin ized under the same condit ions, rechromatographed on Sephadex, f lash ev-aporated to dryness, then resuspended in a minimal amount of water and applied as a long band to Whatman #1 f i l t e r paper and run as a descending chromatogram for about 18 hours in butanol :acet ic acid:water (60:15:25). Under these conditions the DDPS l a b e l conferred extreme mobi l i ty on the labe l led peptide so that the orange band ran just behind the f ront , sep-arating well from the non - labe l led , contaminating peptides which moved only s l i g h t l y due to the i r greater a f f i n i t y fo r the polar , stationary phase. The orange band was eluted with water from the paper. The same procedure was used fo r type B and E peptides.. How-ever, the procedure was changed very s l i g h t l y for preparation of a pep-t ide from A tox in . The suspending f l u i d for l a b e l l i n g with DDPM was changed to 0.05 M guanidine acetate solut ion at pH 7.0 and the excess DDPM was dialysed free from the reaction mixture using M/15 c i t r a t e -phosphate buffer at pH 5 . 6 . The pH was raised to 7.5 with 0.1 N NaOH for subsequent t r yps in i za t ion . If more than one orange band was. ever detected during column chromatography of the t r y p t i c d igest , they were pooled for redigestion 23' and one band was always obtained upon rechromatographing. A quantitative amino acid analysis was c a r r i e d out on each DDPS-peptide. The results, are shown i n f i g u r e V. A s i g n i f i c a n t l o s s of material i s incurred upon preparation of the l a b e l l e d peptides. This l o s s was most severe when working with type A t o x i n , which i s known to undergo aggregation more e a s i l y than types B and E. Much of the protein was l o s t both during the l a b e l l i n g procedure and during d i a l y s i s , when an orange, i n s o l u b l e p r e c i p i t a t e formed. This forced a search f o r a better method f o r preparation of a DDPS-A-peptide. The guanidine-acetate s o l u t i o n was used i n an e f f o r t to break inter-molecular hydrogen bonding and so eliminate aggregation during l a b e l l i n g . To minimize the formation of any more i n s o l u b l e p r e c i p i t a t e upon d i a l y s i s , the d i s t i l l e d water was replaced by citrate-phosphate buffer at pH 5.6, because type A i s most soluble at that hydrogen ion concentration. Upon r a i s i n g the pH with 0.1 N NaOH a very small amount of the i n s o l u b l e material was formed, f a r l e s s than was. observed when using the unmodified procedure. Water was then found s u i t a b l e f o r e l u -t i o n of the t r y p t i c digest through Sephadex as. the danger of aggregation had, by then, been eliminated. I t may even be adviseable i n future to use the modified procedure f o r preparation of the DDPS-peptides of B and E toxins as i t may give more complete l a b e l l i n g and eliminate any small degree of aggregation which may occur. The f i n a l separation of the l a b e l l e d peptide from contaminating ninhydrin-positive peptides on paper also accounts f o r some l o s s of 24 FIGURE V: Representative amino acid analyses of DDPB-peptides; from types A, B and E toxins aspartic glutamic Toxin lysine acid serine acid glycine alanine A jjMoles 0.015 0.023 0.065 0.043 0.066 0.026 Molar ratio 0.70 1.07 3.04 2.00 3.10 1.24 Probable no. of residues 1 1 3 2 3; 1 N-terminal alanine B jjMoles 0.021 0.023 0.056 0.041 0.048 0.037 Molar' ratio 1.05 1.15 2.80 2.00 2.40 1.85 Probable no. of residues 1 1 3 2 2 2 N-terminal glycine E juMoles 0.043 0.039 0.132 0.052 0.136 0.03? Molar ratio 0.83 0.74 2.54 1.00 2.70 0.71 Probable no. of residues 1 1 3 1 3 1 N-terminal glycine 25 sample> considering that about 10% of a sample i s l o s t on one dimensional paper chromatography and that up to another 10% may be l o s t during elut ion from the paper. From one l i t e r of crude toxic f i l t r a t e , type A produces about 1'JJM of pur i f ied tox in , type B about 3 juM and type E about 2 JJM (based on molecular weights of 1 5 , 0 0 0 , 1 0 , 0 0 0 and 15 ,000 respect i ve ly ) . These are only estimations in that they represent the "average" y i e l d of each toxin while the qual i ty of each l i t e r of crude toxin harvested varies tremen-dously. . It i s reasonable to assume about a 66% y ie ld of labe l led pep-t ide from a solut ion of unlabelled tox in , or, 3 juM of toxin w i l l y ie ld about 2 JJM of peptide. Rarely, a second orange band was barely detectable even af ter the second t r yps in i za t ion . Any such contaminant, running ei ther before or a f te r the heaviest band l a t e r subjected to paper chromatography, was discarded. In order to ca lcu late molar ra t ios from the amino acid analyses of the DDPS-peptides, glutamic acid was chosen as e i ther 1 . 0 0 or 2 . 0 0 residues per molecule as i t i s the most stable of the s ix amino acids under condit ions of acid hydro lys is . The only residue number per molecule which was d i f f i c u l t to assess was the number of serine residues present in type E . Although a molar ra t io of 2 .54 i s nearer 3 than 2, i t was more due to the fact that the other two DDPS-peptides each had three serine residues that type £ was assigned three as w e l l . Later sequence work confirmed t h i s . 26 DDPS-cysteine was extensively broken down, even under l imi ted hydrolysis and was, therefore, not ca lcu lab le . However because there i s only one cysteine present in the intact tox ic molecule, there could hardly be more than one in the labe l led peptide. The breakdown products are eluted f i r s t before the ac id i c and neutral amino ac ids . Considering that there i s no tryptophan in types A or B toxins, and therefore none in the labe l led peptides, i t was: assumed that there was none in the peptide obtained from type E e i ther . However, a t rypto -phan determination has yet to be done on type E tox in . S imi la r l y , no methionine i s thought to ex ist in the peptides. Even though methionine breaks down somewhat on hydrolys is , there was none ca lcu lable on the quan-t i t a t i v e amino acid analyses and none detected during the invest igat ion of the amide contents of the peptides. The remarkable s i m i l a r i t y between the analyses of the three DDPS-peptides may.,, therefore, be taken as' addi t ional data in support of the suggestion that these cysteine-containing peptides form a part of the postulated common toxic region of each prote in . 27 Sequence s tud ies : The fol lowing techniques were used to elucidate the sequence of. a l l three peptides: 1. Edman degradation on the in tact DDPS-peptide to obtain the s e -quence near the N - terminal . 2. Ident i f icat ion of the carboxy-terminal using carboxypeptidase B. 3. Cleavage of the intact? dansylated peptide using acid hydrolysis followed by separation of and t o t a l amino acid analyses on the the resul t ing peptides. 4. Cleavage of the i n t a c t , dansylated peptide using s u b t i l i s i n followed by separation of and t o t a l amino acid analyses on the resu l t ing peptides. 5. Appl icat ion of technique #1 on a suitable peptide obtained from s u b t i l i s i n cleavage. In a l l cases, i d e n t i f i c a t i o n of amino acids was accomplished using thin layer chromatography. Technique #1: Sequences at the N - terminal were established using the method of Black (1966), employing the combination of Edman degradations and i d e n t i f i c a t i o n of N - terminal amino acids using thin layer chromatography of dansylated, hydrolysed al iquots of the o r i g i n a l sample as diagrammed in f igure VI. Technique #2: Carboxypeptidase B (Worthington), s p e c i f i c fo r l ys ine and arginine, was suspended in 10% L i C l to a concentration of 10-25 ug/ml 28 FIGURE VI: Sequence studies using technique #1 A DDPS-peptide in h^G^pyridine ( 1 :2) Is 0.3 ml of PTIC* (5% in pyridine) added and incubated 3 hours at 37 C dried down and 0.1 ml TFA* added and incubated at 20 C for 1.5 hours dried down and resuspended in H^O:pyridine (1:2) 25 A removed and 2o"Xof 0.1 M iMaHC03 added dried down and 20 A each of h^ O and DNS-C1* (2.5 mg/ml in acetone) was added. Incubated' 3-4 hours at 37 C dried down and resuspended in 6N HC1 and hydrolyzed identification of the N-terminal amino acid on thin layer plates -DDPS-peptide (residues 2-n) to repeat the above A as B above to obtain -DDPS-peptide (x-n) in h^O:pyridine (1:2) A -=js—as A above to obtain i d e n t i f i -cation of residue 2 -y- as A above to obtain i d e n t i f i -cation of residue x 'Where PTIC (obtained from Dr. John Black, biochemistry department, U.B.C.) stands for phenylisothiocyanate. DNS-C1 (Aldrich Chemical Co. Inc.) stands for l-dimethylaminonaphthalene-5-sulfonyl chloride TFA (Eastman Organic Chemicals) stands-, for trifluoroacetic acid 29 and 0.1 ml was added to an aqueous solut ion of DDPS-peptide at pH 7.6 (Folk et a l . , 1960). At 30 minutes and 1, 2 and 3 hour in te rva l s , a l i -quots were removed, dansylated and run on thin layer p la tes . Technique #3: About 2 uM of the in tac t , dansylated peptide was f lash-evaporated to dryness, resuspended in concentrated HC1 and incubated at 37 C for 40 hours. The hydrolysate was then f lash-evaporated to dryness and resuspended in a minimal amount of water:acetone (1 :1) . Technique #4: About 2 juM of the intact, - , dansylated peptide was f lash-evaporated to dryness and resuspended in 0.10 M NaOAc at pH 7.5 (Sinn et a l . , 1959). The pH had to be adjusted with 1 M acet ic acid to neutral ize the bicarbon-ate remaining from dansylat ion. Then 1% s u b t i l i s i n (N.B.Co.) was added and the solut ion was incubated at 37 C fo r 20 hours. The pH did not r e -quire further adjustment as i t dropped only s l i g h t l y during incubation. Determination of amide content (type B toxin only ) : Intact, undansylated DDPS-B peptide was t o t a l l y degraded with leucine aminopeptidase (LAP) according to the method of H i l l et a l . (1962), omitting the use of pro l idase . The digestion was allowed to proceed fo r IB hours. Glutamic ac id , aspart ic ac id , glutamine and asparagine were run as standards on thin layer plates along with the dansylated d igest . Separation of peptides: In a l l cases electrophoresis followed by ascending chromatography 30 using butanol :acet ic acid:water (60:15:25) was used. Electrophoresis was carr ied out on Watman #3 paper in a formate-acetate buffer (1.5 M formate: 2i".M acetate) at pH 2.0 using 840 volts and 40 ma for 2.3 hours. The yellow, absorbing and f luorescent spots were c i r c l e d af ter each dimension. After the second dimension was completed and the peptides cut out, the chromat-ogram was . f ina l l y sprayed with.ninhydrin (1% in acetone). Thin layer chromatography: a/ Solvents used: For the ac id i c amino acids (cysteine, se r ine , raspar t i c acid and glutamic acid) a polar solvent comprised of propanol:ammonium hydroxide (80:20) was used. For the non-polar amino acids (glycine, alanine and lysine) a non-polar solvent comprised of chloroform:methanol:acetate (95: 10:1) was used. For i d e n t i f i c a t i o n of the t o t a l amino acids present in a given peptide, the hydrolysed and dansylated sample was halved and each half run in a d i f fe rent solvent . b/ Preparation of amino acid standards: 10 JJM of D1MS-C1 in 1.0 ml of acetone and 10 juM of the amino acid in 1.0 ml of 0.1 M NaHCO^ were mixed and l e f t overnight at 20 C in the dark. Then 8 ml of acetone were added and the prec ip i tate so formed was discarded and the supernatant was then stored in stoppered tubes in the dark. 31 esults have been recorded using the fol lowing notat ions: A l l chromatograms are represented as-, l abe l led below: cathode f i r s t dimension anode (electrophoresis) A second dimension (ascending in butanols acet ic acid:water) where: X marks the or ig in where the spot was applied . . . represents the solvent front a f te r ascending :...."> represents the n inhydr in -posi t ive spot O r e presents the f luorescent or yellow spot marked "F" or "Y" respect ive ly A l l peptides are numbered from l e f t to r ight (ninhydrin-pos i t i ve spots omitted). Thus, (^2F^ indicates that the peptide i s f luorescent and posit ioned as indicated in the above hypothetical example. Sequencing r e s u l t s have been presented as fol lows: (1 ,2,3,4) represents the amino acids present with no establ ished sequence 1 -2 -3 -4 represents a known sequence Thus, 1 -2 - (3 ,4) indicates that only the f i r s t two residues' sequence i s establ ished while residues 3 and 4 are known to be present as we l l . 32 Type A DDPS-peptide; Technique #1: a l a - g l u - s e r - c y s - ( 2 s e r , a s p , g l u , 3 g l y , l y s ) Technique #2: l ys P a r t i a l sequence: a l a - g l u - s e r - c y s - ( 2 s e r , a s p , g l u , 3 g l y ) - l y s Technique #3: peptide # content 1 l y s , g l y 2 l y s , g l y , g l u , s e r 3 l y s , g l y , g l u , s e r , c y s , a s p 4 a l a , g l u , s e r P a r t i a l sequence: a l a - g l u - s e r - c y s - ( 2 s e r » a s p » g l u » 2 g l y ) - g l y - l y s Technique if A: peptide # content 1 l y s , g l y , g l u 2 l y s , g l y , g l u , s e r 3 l y s , g l y , g l u , s e r , a s p 4' l y s , g l y , g l u , ser ,asp,cys 5 a l a , g l u , s s r P a r t i a l sequence: a l a - g l u - s e r - c y s - ( 2 s e r , a s p , g l u , 2 g l y ) - g l y - l y s Technique #1 applied to s u b t i l i s i n peptide #: 1: g l u - g l y - g l y - g l y - l y s . 4: cys - se r -asp -se r Total sequence: 1 2 3 4 5 6 7 8 9 10 11 12 a l a - g l u - s e r - c y s - s e r - a s p - s e r - g l u - g l y - g l y - g l y - l y s 33 Type B DDPS-peptide: Technique #1: gly-ala-glu-ser-(cys,2ser,asp,glu,ala,gly,lys) Technique §2 : lys Partial sequence: g ly -a la -g lu-ser- (cys»2ser,asp,g lu,a la,g ly ) - lys Technique #3: peptide # content 1 lys,gly 2 lys,gly,ala 3 lys,gly,ala,glu,ser 4 lys,gly,ala,glu,ser,asp,cys Partial sequence: gly-ala-glu-ser-(cys,2ser,asp,glu)-ala-gly-lys Technique #4 peptide § content 1 lys, gly,ala, glu 2 lys,gly,ala,glu,ser 3 lys,gly,ala,glu,ser,asp 4 lys,gly,ala,glu,ser,asp,cys 5 gly» ala,glu,ser Partial sequence: gly-ala-glu-ser-(cys,ser,asp)-ser-glu-ala-gly-lys Technique #1 applied to subtil isin peptide #: 4: cys-ser-asp-(ser,glu,ala,gly,lys) Total sequence: 34 Type E DDPS-peptide: Technique #1: gly-ala-glu-ser-(cys,2ser,asp,2gly,lys) Technique #2: lys Partial sequence: gly-ala-glu-ser-(cys,2ser,asp,2gly)-lys Techniaue //-3: peptide// content 1 lys,gly 2 lys,gly, ser,asp, cys 3 lys,gly, ser,asp, cys 4 lys,gly, ser,asp, cys,glu 5 gly,ala, glu,ser Partial sequence: gly-ala-glu-ser-(cys,2ser,asp,gly)-gly-lys Technique #4 content lys,gly,ser glu,ser,cys,asp lys,gly,ser,asp,cys lys,gly,ser,asp,cys,glu gly,ala,glu,ser Partial sequence: gly-ala-glu-ser-(cys,2ser,asp,gly)-gly-lys Technique #1 applied to subti l is in peptides #: 1: ser-gly-gly-lys 2: cys-ser-asp-(ser,glu) Total sequence: 1 2 3 4 5 6 7 8 9 10 11 gly-ala-glu-ser-cys-ser-asp-ser-gly-gly-lys 35 Amide estimation Using enzymatic digestion of intact DDPS-B peptide, the aspartic and glutamic acid residues were' found to be in the amide form. No trace of the dicarboxylic acid forms was detected. It is assumed that the A and E peptides are similar in their amide content. The 3 DDPS-peptides may now be. written in the following way to show the common sequence of seven residues: A ala-glu-ser-cys-ser--asp-ser -NH_ NH„ 2 2 B -ala-glu-ser-cys-ser--asp-ser-NH_ NH_ 2 2 E g!y' -ala-glu-ser-cys-ser--asp-ser-• NH_ NH_ 2 2 glu-gly-gly-lys NH2 glu-ala-gly-lys-NH2 gly-gly-lys No tryptophan determination has. been done on the E peptide but it is known that there is no tryptophan in either A or B toxins. 3 6 The intact DDPS-peptide was dansylated before fragmentation in order to provide a t o t a l of three "markers". That i s , a f te r hydrolysis any peptide containing the o r i g i n a l N- or C-terminals or the DDPS-labelled. cysteine could be located on the chromatogram. Considering that the i n -tact peptide was rather short (11-12 residues) there was a good chance of obtaining most of the peptides released af te r hydro ly is . The two-dimensional chromatograms were always done twice. The f i r s t time T of the mater ia l was spotted. • In th is way, two spots could sometimes be detected which would have appeared as one i f a greater conc-entration were used. On the other hand, when the remaining - j - were spot -ted, a spot would sometimes be detected which was, missed on the f i r s t chromatogram. Although th is procedure was obviously wasteful , i t afforded the best r e s u l t s . The diagrammed chromatographic resu l ts represent a l l the peptides obtained combined on one diagram, although two separations were carr ied out as descr ibed. Amino acid analyses were done on pooled, eluted peptides from the two chromatograms,. No peptide could be obtained from acid hydrolyis in s u f f i c i e n t quantity for sequencing or even for an N-terminal . There was s u f f i c i e n t material in each case to f ind only t o t a l amino acids by running hal f of the sample in each solvent as previously described. However, s u b t i l i s i n afforded a much better y i e l d . The peptide chosen from th is procedure was one that was present in high concentration and which probably represented that portion of the in tact peptide s t i l l not sequenced. The choice was guided by the t o t a l amino acid analysis done on small a l iquots of each of the pooled, eluted peptides. 3 7 Another serious l o s s i n material was afforded when poor separ-ation of. fragments of the i n t a c t DDPS-peptide was obtained. Occasionally, two peptides would appear touching one another or even as. one continuous "smeared" spot on the chromatogram. To minimize contamination of one peptide with another only of each spot was eluted, leaving the touching halves on the chromatogram. Thus, i n one step, 50% of such a peptide had to be discarded as contaminated. In theory the Edman degradation, e s p e c i a l l y as i t was used here, could be used to sequence the whole peptide with j u s t one sample. How-ever, i n pr a c t i c e the small losses i n material at each step are enough to make i t very d i f f i c u l t to obtain more than 4 residues from the IM-terminal when s t a r t i n g with only about 1 p?4 of l a b e l l e d peptide. I t was also noted that the DDPS-label may have conferred s u f f i c i e n t non-polar character to the l a b e l l e d i n t a c t peptide to cause i t to become s l i g h t l y soluble i n the benzene used to extract the reaction mixture a f t e r formation of the phenylthiocarbamyl peptide. I t was d i f f i c u l t to accurately assess t h i s l o s s but i t may account, i n part, f o r the i n a b i l i t y to obtain more information from the i n i t i a l 1 p.M of sample. Rather than simply applying technique #1 to more mate r i a l , i t was thought that the use of other techniques along with the f i r s t would be not only more e f f i c i e n t , but would also serve as a means of constantly checking the data obtained. That i s , an i n c o r r e c t i n t e r p r e t a t i o n of a th i n l a y e r p l a t e , f o r example, would be detected when the data obtained using a d i f f e r e n t technique did not agree. I t i s estimated that about 3 JJM of each DDPS-peptide were used 38 to elucidate i ts sequence. The total sequence of each peptide was always based, then, on the correlation of data obtained from more than one technique. Also, any doubtful portion of any sequence was checked by eluting an appropriate peptide obtained from the subtilisin digest for an IM-terminal analysis or for more extensive use of technique #1. Results obtained in this way were not specially noted in the results as they served only to confirm the data already recorded. Where a peptide was thought to contain a repeating residue (ie. gly-gly-) i t was sequenced using technique //l synchronously with a second peptide so as to check that the Edman degre-dation was proceding normally. Carboxypeptidase was. not used to digest the peptide for the amide estimation as the hydrolysis of peptide bonds involving dicarboxylic amino acids is very slow (Canfield and Anfinsen, 1963). When glutamine and asparagine appear several times in a peptide the conversion of the amides to the free carboxyl-forms is known to occur, but as no trace of either dicarboxylic acid was seen, i t was concluded that each was present in the amide form. 39 Inactivation experiments using acetylcholine (ACH): Only B Lamanna toxin was used. Three tubes were used as fo l l o w s : 1 2 3 Toxin (1 mg/ml) 3 ml 3 ml ' -ACH (10 mg/ml) 0.1 ml ' - 0.1 ml Saline - 0.1 ml 3 ml The toxin was; suspended i n citrate-phosphate buffer at pH 5.6 A l l three tubes were incubated f o r 4 hours. At 1, 2, 3 and 4 hour i n t e r v a l s 0.1 ml were removed from each tube, d i l u t e d appropriately and t i t r a t e d . The remaining 2.7 ml i n each tube was dried down and spotted on Watman #1 f i l t e r paper along with choline and acetylcholine standards. The chromatogram was ascended f o r about 3 hours (in propanol: ll\l NaOAc (1:1) saturated with EtOAc) to a t t a i n about an 8" r i s e . Detec-t i o n of spots was accomplished using 1% I^ i n MeOH. With t h i s system, ACH runs about half-way between the o r i g i n and the front and choline appears j u s t s l i g h t l y more retarded but well-separated from the ACH. There was no detection of e i t h e r a decrease i n t o x i c i t y or the ap-pearance of choline i n the reaction mixture a f t e r incubation. The mode of action of the botulinus toxins i s s t i l l unknown although i t has been postulated that they prevent the release of ACH at the myoneural junction. However, evidence f o r t h i s hypothesis i s based on- unconvincing electron micrographs and no d e f i n i t i v e chemical work has been done on a pure toxin i n terms of i t s reaction mechanism. 40 It is known that poisoning results, in flaccid paralysis, or, inhibition of neural transmission. The fact that paralysis is f i r s t evident at the extremities probably has'a purely physiological explana-tion, for i t is known that introduction of the toxin intravenously rather than intraperitoneally or through the gastrointestinal tract causes death in the mouse within 15 minutes. Another fact to consider is that very few toxin molecules are required to effect death: once ca l -culated to be about lO*^ molecules for a man of average body weight. Taken together these two facts suggested that the toxin may act enzymatically to break down ACH after i t ' s release. One could reas-onably postulate a thioester intermediate analagous to that proposed for papain-catalyzed reactions (Durell and Fruton, 1954): f « 3 ac&fy/cAo//n* Ctjj i40-fcH a) 2-j4-C^ choline. c S H •= .s - 5 •t-ax./n \ I | It was thought that such a reaction mechanism, though incomplete as i t is written here, may explain the deactivation that occurs when the toxins are exposed to an alkaline pH. Therefore, an experiment was carried out to either prove or disprove this reaction with the view in mind being 41 that the results would be valuable, whether positive or negative. The dif f icult ies in studying the mode of action of the toxins are, at present, insurmountable. Prerequisite to such studies are: a/ " . . .a precise knowledge of the chemical nature of the functional groups and...of the spatial relation of every atom with respect to a l l other atoms." (Hofmann, 1963.) Sequence studies on the intact type. B toxin are just beginning. b/ the identification of the substrate, or, moiety affected by the toxins. With regard to the latter, there is s t i l l no proof that the toxins act enzymatically. It is possible that they induce the produc-tion of some other molecular species in_ vivo in order to disrupt neural transmission. Or, the toxins may bind with some receptor sites or with some essential molecular species in order to cause an inactivation (cf. the inhibition of ACH release). It is also possible that they may in some way alter an electron transport system essential for transmission. Not only is the pharmacological mode of action of the toxins unknown, the exact role they play in the bacterial ce l l remains a mystery. 42 GENERAL DISCUSSION Perhaps the results presented here would be more significant when considered in the light of similar work carried out on other pro-teins containing reactive sulfhydryls which have been established as being involved in active sites. An excellent review by Cecil (1963) l isted only four such pro-teins, as previously reviewed. Indeed, in only three of these cases had a cysteine-containing peptide been characterized, the evidence in the case of papain being based on reaction kinetics. It was also reported that the SH groups formed S-acyl derivatives and that there was no case of an SH group reacting in any other way. Evidence for the involvement of cysteine in the active site of the triosephosphate dehydrogenases: was also supplied by the finding that the thiol-containing peptides obtained from rabbit and pig muscle and yeast dehydrogenases were identical. Yet, only one year later, Harris (1964) and Lee et a l . (1964) showed that alcohol dehydrogenase obtained from horse l iver and from yeast contained thiol peptides of a striking functional relationship. And, in April 1965, Fondy et a l . reported similar.findings when comparing lactate dehydrogenases from diverse sources. Although the cysteine res-idues of the dehydrogenases just mentioned have not been shown to form thiol esters, the isolation of such peptides would indicate that they may form the active sites of their respective tetramers. As previously reviewed, the thiol peptides isolated from alco-hol and lactate dehydrogenases would appear to represent the coenzyme-43 binding s i t e s while the s i t e of the triosephosphate dehydrogenase l a b -e l l e d by Harris (1964) i s probably the substrate-binding s i t e . Fondy and his coworkers have speculated that i f the coenzyme-binding region of the triosephosphate dehydrogenase does involve t h i o l groups i n a manner comparable to the l a c t i c and alcohol dehydrogenases, then any r e l a t i o n -ship among t h e i r coenzyme-binding s i t e s could center i n the second, non-alkyla t e d cysteine i n the e s s e n t i a l t h i o l peptide of the triosephosphate dehydrogenase (the l a t t e r enzyme from yeast having only two -SH groups per subunit). Therefore, by matching the l a b e l l e d cysteine residues i n the former dehydrogenases with the unlabelled cysteine i n the t h i o l pep-t i d e of the triosephosphate dehydrogenase, one sees that a f u n c t i o n a l s i m i l a r i t y may e x i s t among the coenzymer-binding regions of a l l three dehydrogenases (see appendix). In view of the above, i t i s i n t e r e s t i n g that i n addition to binding the i d e n t i c a l coenzymes and functioning by means of e s s e n t i a l t h i o l groups, many of the dehydrogenases show measureable c r o s s - s p e c i f i c -i t y f o r one another's primary substrates (Struck et a l . , I960; Fisher et a l . , 1961;- Tomkins et a l . , 1961; Fishbein et a l . , 1964; and Shaw et a l . , 1964). Perhaps some of the s i m i l a r i t i e s and the differences among the e s s e n t i a l t h i o l peptides, of the various pyridine nucleotide dehydrogenases w i l l provide some clues to the mechanism of coenzyme binding and of substrate s p e c i f i c i t y . Considering b i o l o g i c a l l y a c t i v e proteins i n general (rather than -SH enzymes i n p a r t i c u l a r ) , i n the l a s t 2-3 years the presence of 44 similar amino acid sequences has been established in the active site regions of functionally similar proteins. These are l isted in the appen-. dix. The esterases aliesterase, pseudocholinesterase, trypsin and chymo-trypsin a l l contain a pentapeptide (in which is found the active serine residue) which is also found in E.coli alkaline phosphatase. Papain and f i c in , two plant proteases, are also very similar in function and i t is not suprising, therefore, that they should yield nearly identical peptides. Also included are some of the polypeptide hormones which have been a source of evidence completely in support of the concept that a. sequence of amino acids common to functionally identical proteins (or peptides) is probably the active site. A good example is adrenocortico-tropin (ACTH) which has a total of 3:9 residues and demonstrates species differences between residues 25-32. In 1962 (a, b) Hofmann synthesized an eicosapeptide amide corresponding to the sequence of the N-terminal 20 residues to obtain a very active peptide hormone. Also a good-example are the melanocyte-expanding peptide hormones ( -MSH and |3 -MSH as well as ACTH) a l l of which contain a common heptapeptide sequence. Interest-ingly, this sequence is found in a porcine l ipolyt ic peptide investigated by Astwood et a l . (1961) which also has marked MSH activity. Included in the appendix are two chemically synthesized peptides which also exhibit MSH activity. Witter and Tuppy's work (1960) on serum albumins from different sources should be mentioned here. Their DDPM-labelling and fragmentation of two different serum albumins yielded two identical peptides. The 45 exact role of these peptides in the intact molecules is s t i l l not es-tablished. The significance of such similarities in sequence was; noted by Canfield and Anfinsen (1963) who referred to a peptide obtained from cytochrome c which was shown to vary relatively l i t t l e in amino acid sequence over a wide range of phyla. These authors pointed out that: "These findings have suggested the functional importance of that (common) portion...since considerably greater variations in structure might have been expected (perhaps with some naivete) as the result of mutational events during evolution were such events not detrimental to the organism (or of no selective advantage) This study is in every way analogous to the comparative work cited on functionally identical proteins obtained from diverse sources. Although the production of botulinus toxins is confined to one bacterial species, their lack of similarity has been established by their dis-tinct immunological characteristics, their molecular weights, electro-phoretic mobilities and their amino acid contents (Gerwing et a l . , 1964,-1965a, 1965b, and 1966; Van Alstyne et a l . , 1966). At the same time, types A-F a l l appear to have identical functions which inspire the con-cept of a common, toxic s ite. The active site of no other toxin has yet been elucidated. Consequently the data presented here are a l l the more significant not only because they correlate w.ell with similar work on plant and animal proteases and dehydrogenases, but also because they are derived from a unique source. 46: CONCLUSIONS It has been shewn that: 1/ Like types B and E, type A toxin contains a single cysteine residue. The total amino acid analysis indicated a minimum molecular weight of 15,000 which can also be calculated for the crystal-line toxin used by Buehler et a l . as discussed. No tryptophan was detected, conflicting with the contention of Boroff and co-workers that tryptophan is implicated in the active region of botulinus toxins. 2/ A l l three toxins are inactivated to a significant degree by iodo-acetate and by two compounds highly specific for the sulfhydryl group of cysteine, PCMB and DDPM. Studies with PCMB showed good correlation between loss of ac-t iv i ty and moles of mercaptide formed. 3/ One peptide labelled with DDPM was obtained from each toxin using gel f i l t rat ion through Sephadex G-25 and then paper chromatography after exhaustive trypsinization. 4/ The amino acid analyses of the three DDPS-peptides are remarkably similar: . lys asp ser glu gly ala type A 1 1 3 2 3 1 type B . 1 1 3 2 2 2 . . type E 1 1 3 1 3 1 47 5/ The three DDPS-peptides contain a common sequence seven residues in length: type A •ala-gln-ser-cys-ser-asn-ser4gln-gly~gly-gly-lys type B glyTala-gln-ser-cys-ser-asn-ser4gln-ala-gly-lys type E gly-j-ala-gln-ser-cys-ser-asn-ser-igly-gly-lys 6/ Type B botulinus toxin does not act enzymatically to alter acetyl-choline and thus effect flaccid paralysis. It i s , therefore, suggested that the above thiol peptides contain a l l or part of a postulated active site common to each of types A, B and E botulinus toxins. 48a KEY TO APPENDIX Peptide source and references: Dehydrogenases: a/ Lactic dehydrogenase; Fondy et a l . , 1965. b/ Horse l i v e r alcohol dehydrogenase; Harris, 1964 and Lee et a l . , 1964. c/ Yeast alcohol dehydrogenase; Harris, 1964. d/ Rabbit and pig muscle and yeast triosephosphate dehydrogenase; Harris, 1963 and Perham and Harris, 1963. e/ Same as d/ with the carboxy-terminal written f i r s t . Enzymes with active serine residues; a/ Aliesterase; Jansz et al.., 1959a. b/ Chymotrypsin; Hartley, 1962. c/ Trypsin; Dixon et a l . , 1958. d/ Elastase; Hartley, I960. e/ Pseudocholinesterase; Jansz et a l . , 1959b. f/ Thrombin; Hartley, 1960. g/ E.coli alkaline phosphatase; Schwartz et a l . , 1963. h/ S u b t i l i s i n ; 5anger and Shaw, 1960. Plant proteases: a/ Papain; Light and Smith, 1964 and Light et a l . , 1964. b/ Fi c i n ; Wong and Liener, 1964. Cytochrome c: a/ Beef, horse and pig heme c peptides; Tuppy, 1958. b/ Salmon heme c peptide; Tuppy, 1958. c/ Chicken heme c peptide; Tuppy, 1956. 48b d/ Silkworm heme c peptide; Tuppy, 1958. e/ Yeast heme c peptide; Tuppy? 1958. Rh, rubrum heme c peptide; Tuppy, 1958. Serum albumins: a/ and b/ human and bovin serum albumins; Witter and Tuppy, I960. MSH and ACTH: a/ Pig, beef, horse and monkey o(-M5H; Harris, 1959 and L i , 1959. b/ Pig |§-MSH; Harris and Roos, 1959. c/ Beef !^ -MSH; Geschwind et a l . , 1957. d/ Horse (S-MSH; Dixon and L i , 1961. e/ Monkey -^MSH; Li et a l . , 1961. f/ Human (3-MSH; L i et a l . , 1961. g/ N-terminal 15 residues in ACTH; Hofmann and Katsoyannis, 1963. h/ Pig l ipo lyt ic peptide I; Astwood et a l . , 1961. i / Chemically synthesized octapeptide with marked MSH activity; Hofmann et a l . , 1957. j / Same as i / ; Hofmann et a l . , 1962. Unlesa otherwise indicated, peptides are written with the N-terminal f i r s t . Residues: marked with a " * " are those found to be involved in the active site of the intact protein. 48c APPENDIX Peptides common to functionally similar proteins Dehydrogenases: a/ val-ileu-ser-gly-gly-cys-asn-leu-asp-thr-ala-arg-lys b/ val-ala-thr-gly-ileu-cys-arg-ser-asp-asp-his-val-thr-ser-gly-leu-c/ try-ser-gly-val- cys-his-thr-asp-leu-his-ala-try-his-gly-asp * d/ val-ser-asn-ala-ser- cys-thr-thr-asn-cys-leu-ala-pro-leu-ala-lys e/ lys-ala-leu-pro-ala-leu-cys-asn-thrTrthr-cys-ser-ala-asn-ser-val-ileu-Enzymes wi th active serine residues: a/ gly-glu-ser-ala-gly-gly * b/ sei>-ser-cys-met-gly-asp-ser-gly-gly-pro-leu-val-cys-lys -M. c/ ser-cys-glu-gly-gly-asp-ser-gly-pro-val-cys-ser-gly-lys d/ gly-asp-ser-gly e/ phe^gly-glu-ser-ala-gly jr. f/ gly-asp-ser-gly g/ pro-asp-try-val-thr-asp-ser-ala-ala-ser-ala h/ thr-ser-met-ala Plant proteases: a/ pro-val-lys-asn-gln-gly-ser-cys-gly-ser-cys b/ pro-ileu-arg-glu-glu-gly-glu-cys-gly-ser-cys Cytochrome c: a/ val-gln-lys-cys-ala-gln-cys-his-thr-val-glu-lys b/ val-gln-lys-cys-ala-gln-cys-his-thr-val-glu c/ yal-gln-lys-cys-ser-gln-cys-his-thr-val-glu 48d d/ val-gln-arg-cys-ala-gln-cys-his-thr-val-glu e/ phe-lys-thr-arg-cys-glu-leu-cys-his-thr-val-glu f/ cys-leu-ala-cys-his-thr-phe-asp-glu-gly-ala-asn-lys Serum albumins; a/ leu-glu-asp-glu-gln-glu-cys-pro-phe b/ leu-glu-asp-glu-gln-glu-cys-pro-phe MSH and ACTH; a/ Ac-ser-try-ser-met-glu-his-phe-arg-try-gly-lys-pro-val-N b/ asp-glu-gly-pro-try-lys-met-glu-his-phe-arg-try-gly-ser-pro-pro-lys-asp c/ asp-ser-gly-pro-try-lys-met-glu-his-phe-arg-try-gly-ser-pro-prb-lys-asp d/ asp-glu-gly-pro-try-lys-met-glu-his-phe-arg-try-gly-ser-pro-arg-lys:-asp e/ asp-glu-gly-pro-try-arg-met-glu-his-phe-arg-try-gly-ser-pro-pro-lys-asp f/ asp-glu-gly-pro-try-lys-met-glu-his-phe-arg-try-gly-ser-pro-pro-lys-asp ala-glu-lys-lys g/ ser-try-ser-met-glu-his-phe-arg-try-gly-lys-pro-val-lys-gly h/ asp-glu-gly-pro-try-lys-met-glu-his-phB-arg-try-gly-ser-pro-pro-lys-asp i / ser-met-glu-his-phe-arg-try-gly j / his-phe-arg-try-gly-lys-prorrval BIBLIOGRAPHY Alexander, N.H. 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