UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Purification and characterization of a toxic protein of clostridum botulinum type E, strain Iwanai. Bains, Hardial Singh 1964

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1964_A6_7 B35.pdf [ 5.87MB ]
Metadata
JSON: 831-1.0104867.json
JSON-LD: 831-1.0104867-ld.json
RDF/XML (Pretty): 831-1.0104867-rdf.xml
RDF/JSON: 831-1.0104867-rdf.json
Turtle: 831-1.0104867-turtle.txt
N-Triples: 831-1.0104867-rdf-ntriples.txt
Original Record: 831-1.0104867-source.json
Full Text
831-1.0104867-fulltext.txt
Citation
831-1.0104867.ris

Full Text

PURIFICATION AND CHARACTERIZATION OF A TOXIC PROTEIN OF CLOSTRIDIUM BOTULINUM TYPE E, STRAIN IWANAI by HARDIAL SINGH BAINS B.Sc, Panjab University, 1959 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF M.Sc. in the Department of BACTERIOLOGY AND IMMUNOLOGY We accept t h i s thesis as conforming to the required standard: THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1 9 6 4 In presenting this thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that per-mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publi-cation of this thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission^ Department of j^^h^AA-Ab^f ^A^v^v^d&r^ The University of B r i t i s h Columbia, Vancouver 8, Canada Date /3-S~^^/  i A B S T R A C T Clostridium botulinum type E, s t r a i n Iwanai, was grown i n a dialysate apparatus, modified from the o r i g i n a l apparatus of Vinet and Fredette (1951), using GPB1 medium with 0.5 percent dextrose and 1.0% sodium t h i o g l y c o l l a t e , f o r f i v e days at 30° C. The toxin was precipitated from the c e l l - f r e e toxic f i l t r a t e with 0.60 saturated ammonium sulfa t e at 4° C overnight. The toxic p r e c i p i t a t e was obtained by centrifugation at 4,500 R.P.M. at 0° C for 45 minutes and dissolved i n 0.01 M sodium acetate buffer at pH 5.5. The preparation was dialysed f o r 24 hours against the same buffer i n versene-treated d i a l y s i s sacs and the insoluble material was removed by centrifugation. This preparation was then applied to ion-exchange columns. DEAE (Selectacel) c e l l u l o s e , suspended and kept in 2 M NaCl at 4 G C, was packed into columns under gravity flow at room temperature, washed with IN HC1 and equilibrated with 0.01 M sodium acetate buffer at pH 4.5. The toxin, eluted by the same buffer, reprecipitated and rechromatographed, was further analysed in the ultracentrifuge and with e l e c t r o -phoresis. T o x i c i t y of the pure preparation was found to be 7.5 x 10 MLD/mg N, the sedimentation c o e f f i c i e n t , SggW = 1.70, and the molecular weight 18,600. P a r t i a l amino acid analysis on t h i s preparation was also c a r r i e d out. A C K N O W L E D G E M E N T S I take t h i s opportunity t© thank Dr. J u l i a Gerwing, under whose d i r e c t supervision t h i s work was c a r r i e d out. I have p r o f i t e d a great deal from her able guidance and know-ledge. I am indebted to her also f o r allowing me to work on the problem of the p u r i f i c a t i o n ©f a toxic protein produced by Clostridium botulinum type E which has helped me to understand the problems of protein p u r i f i c a t i o n i n general and ion exchange chromatography in p a r t i c u l a r . I thank Dr. C.E. Dolman fo r placing his confidence in my research c a p a b i l i t i e s and making i t possible f o r me to pursue t h i s course of research. I am grateful to Dr. M. E. Reichmann fo r h i s a s s i s -tance and advice given regarding the biophysical studies c a r r i e d out during the past two years. I appreciate the continuous help given to me by Mr. Angus Macaulay. i i F.Q R E W 0 R- D-Work has been done previously on various botulinus toxic proteins in order to p u r i f y and further characterize these materials. High molecular weights f o r type A, B, and D have been reported before but no attempts had been success-f u l in obtaining botulinus type E in pure form. (Sakaguchi and Sakaguchi, 195 9; Sakaguchi et a l , 1963; Gerwing et a l 1962; Gordon et a l 1957). This toxin i s highly l e t h a l for man and i t s t o x i c i t y i s potentiated by various p r o t e o l y t i c enzymes including trypsin (Dolman 1953, 1957; Sakaguchi and Tohyama, 1955; Duff et a l 1956). In order to study these a c t i v i t i e s , a thorough investigation of the molecular s i z e and character of t h i s material, i s necessary. This report has covered various a n a l y t i c a l techniques in protein p u r i f i -cation and characterization. A toxic preparation has been obtained which appears to be homogeneous by both ultracen-t r i f u g a l and electrophoretic analysis. IV TABLE OF CONTENTS ABSTRACT IForevord' Acknowledgements Table of Contents L i s t of Figures Page Chapter I A n a l y t i c a l methods i n Protein Chemistry (a) Protein structure 1 (b) Isolation 2 (c) Protein s o l u b i l i t y and p r e c i p i t a t i o n 3 (d) Protein p u r i f i c a t i o n 4 1. Repeated P r e c i p i t a t i o n 5 2. Adsorption chromatography 6 3. Ion Exchange Column Chromatography 7 4. Zone electrophoresis 10 (e) C r i t e r i a of Purity 13 1. C r y s t a l l i z a t i o n 13 2. B i o l o g i c a l c r i t e r i a 13 3. Chemical structure 14 4. Chromatography 14 5. Zone electrophoresis 14 6. U l t r a c e n t r i f u g a l analysis 14 7. D i f f u s i o n electrophoresis 15 (f) Amino acid analysis 16 Chapter II Review Literature on Botulinus Proteins 18 (a) Clostridium botulinum type A 19 (b) " " " B 28 (c) " " «» C (C^ and 32 C g ) (d) " " " D 35 (e) " " V E 36 Chapter III Preliminary Experiments (a) Mass production of toxin 39 (b) P u r i f i c a t i o n of concentrated toxin 40 (c) Preparation of soluble concentrated toxin 51 Chapter IV Materials and Methods 59 (a) Toxin production 60 (b) Toxin p r e c i p i t a t i o n 60 (c) Toxin p u r i f i c a t i o n 60 V (d) Treatment of d i a l y s i s paper 61 (e) Assay technique 61 (f) A n a l y t i c a l techniques 61 (g) Preparation of Buffer solutions 62 Chapter V Results 63 (a) Degree of p u r i f i c a t i o n 63 (b) E l u t i o n Chromatography 63 (c) U l t r a - V i o l e t absorption spectrum 63 (d) U l t r a c e n t r i f u g a l analysis 67 (e) Electrophoretic analaysis 67 (f) D i f f u s i o n Properties 67 (g) Molecular Weight 67 (h) Amino Acid Analysis 71 Chapter VI Discussion 73 Summary 73 Appendices 83 References and Bibliography (VI) LIST OF FIGURES F i g . 1 Dialysate apparatus f o r the mass production of botulinus, type E toxin. Figi. 2 Modified dialysate apparatus f o r the mass produc-ti o n of botulinus type E toxin. F i g . 3 Chromatography of botulinus type E, ethanol p r e c i p i t a t e d toxin on a column packed with DEAE pretreated with 2 M sodium acetate solution. The graph shows the el u t i o n pattern.of the preparation. F i g . 4 Separation of a non-toxic and water soluble material from the toxic preparation of botulinus type E with chromatography on DEAE, 2 M NaCl pretreated columns. Elution was c a r r i e d out with d i s t i l l e d H 20. F i g . 5 Stepwise elu t i o n gradient of botulinus type E, ethanol p r e c i p i t a t e d toxin DEAE Cellulose (Selectacel) columns, pretreated with 2 M NaCl at 4 ° C, with d i s t i l l e d HO and 1M sodium acetate solution at pH 6.5. 2 F i g . 6 Stepwise e l u t i o n gradient of botulinus type E, ethanol p r e c i p i t a t e d toxin, using 1M sodium acetate solution at pH 6.5, pH 4.5 and pH 7.0 respectively. Water soluble non-toxic material i s previously removed with d i s t i l l e d H 20 (DEAE-Cellulose i n 2 M NaCl i s used in the column). F i g . 7 Stepwise elu t i o n gradient of botulinus type E, ethanol p r e c i p i t a t e d toxin. (1M sodium acetate solution at pH 6.5 and pH 4.5, i s used as eluent. The column i s packed with DEAE Cellulose, pretreated with 2M NaCl at 40 C) . F i g . 8 Chromatography of botulinus type E, ethanol p r e c i p i t a t e d toxin on a column packed with Dowex-50, pretreated with 2M NaCl. E l u t i o n was c a r r i e d out with d i s t i l l e d and 1M sodium acetate solutions (pH 6.5 and pH 4.5 re s p e c t i v e l y ) . (VII) F i g . 9 Chromatography of botulinus type E, ethanol prec i p i t a t e d toxin on DEAE c e l l u l o s e column, pretreated with 2M NaCl. The column i s washed with IN HC1 and d i s t i l l e d water a f t e r packing, and stepwise elution was c a r r i e d out with d i s t i l l e d H o0 and 0.05 M and 0.1 M sodium acetate solutions at pH 6.5 respectively. Fig.10 Schlieren photograph of u l t r a c e n t r i f u g a l behaviour of botulinus type E, ethanol pre c i p i t a t e d and p u r i f i e d toxin. Fig.11(a) Chromatography of botulinus, type E, (NH) SO 4 2 4 p r e c i p i t a t e d toxin on a DEAE c e l l u l o s e column. Fig.11(b) Same as Fig.11(a) but the graph i s plotted using a d i f f e r e n t scale. Fig.12 Chromatography of botulinus, type E, (NH 4> 2S0 4 p r e c i p i t a t e d toxin on a DEAE c e l l u l o s e column. Fig.13 Chromatography of botulinus type E, (NH.) SO p r e c i p i t a t e d toxin on a DEAE c e l l u l o s e column, pretreated with 2M NaCl, washed with IN HC1; equilibrated and eluted with 0.01 M sodium acetate buffer at pH 4.5. Fig.14 Chromatography of botulinus type E (NH 4> 2S0 4 p r e c i p i t a t e d toxin (same conditions as in F i g . 13). This figure shows the elution of the adsorbed non-toxic material with IN HC1 from the column afte r elution of the toxin. Fig.15 Rechromatography of botulinus type E, chromatographed and reprecipitated toxin. The figure shows a single sharp peak. Fig.16 U l t r a - v i o l e t absorption spectrum of pure botulinus type E toxin. Fig.17 U l t r a c e n t r i f u g a l analysis of botulinus type E, p u r i f i e d toxin. Schlieren photographs were taken at sixteen minute i n t e r v a l s . Ultracentrifuge was run at 59780 RPM. VIII F i g . 18 F i g . 19 F i g . 20 F i g . 21 F i g . 22 Electrophoretic behaviorocS: botulinus type E, p u r i f i e d toxin. Comparative chromatographic behavior of dialysate and f l a s k toxins of botulinus type E, p r e c i p i t a t e d and eluted under s i m i l a r conditions. Chromatographic behavior of botulinus type E, (NEL) SO p r e c i p i t a t e d toxin. Changes in pH during the treatment of DEAE Cellulose (OH~form) with 1M NaCl. Electro-chromatographic apparatus. (1) CHAPTER I An a l y t i c a l Methods in Protein Chemistry The synthetic production of urea from an inorganic source (ammonium cyanate) by WShler i n 1828, was the "beginning of the end" of an era infested with superstition and misinterpretation of s c i e n t i f i c l o g i c . A l i t t l e more than a century l a t e r another important stage in the develop-ment of molecular biology was marked by the i s o l a t i o n and c r y s t a l l i z a t i o n of the Tobacco Mosaic Virus by Stanley. During t h i s period a number of organic compounds were is o l a t e d and t h e i r b i o l o g i c a l a c t i v i t y studied. Two decades were yet to pass before techniques were-developed s u f f i c i e n t l y to allow the st r u c t u r a l studies on macromolecules of b i o l o g i c a l o r i g i n . In 1945 Sanger reported the complete amino acid sequence of the i n s u l i n molecule. Watson and Crick followed him by proposing the molecular structure of genetic material (DNA and RNA). The major advances made i n the study of molecular biology during the past t h i r t y years are a di r e c t r e f l e c t i o n on the techniques made available to the researcher during t h i s time. The following pages deal b r i e f l y with the pr i n c i p l e s involved in the i s o l a t i o n and characterization of proteins. (2) (a) Structure of Proteins Proteins are giant molecules with great d i v e r s i t y and complexity i n structure; however, the basic structure of a l l proteins i s the same i n that they consist of long chains of amino acids. There are twenty L-amino acids, of common occurrence. The amino acids, with t h e i r dipolar ion groups form the -NH-CH-CO- group with the elimination of water during polymerization. The twenty amino acids have a d i f f e r e n t R-group or side chain which i s s p e c i f i c f o r each amino acid. A peptide linkage of a l l or some of these combinations imparts to polypeptides t h e i r s p e c i f i c a c t i v i t y . There may be one or more polypeptide chains which may i n turn consist of several thousand amino acid residues, that com-prise a protein. The amino acids of the polypeptide are arranged in a genetically determined d e f i n i t e sequence which comprises the primary structure of proteins. Long chain polymers, with t h e i r apparent tendency for a screw symmetry, tend to be dependent on th e i r side chains f o r establishing s t a b i l i t y . No configuration i s stable unless i t allows every Imino-group to be hydrogen-bonded to a carbonyl belonging to the same chain or to a neighboring one. There are various ways i n which hydrogen bonding occurs i n polypeptide chains. The simplest i s a planar zig-zag chain which occurs i n synthetic polyamide (nylon). The other types of structures with hydrogen bonds occur within the same (3) chain and are designated as either alpha-or b e t a - h e l i c a l . The X-ray d i f f r a c t i o n photographs of c r y s t a l l i n e proteins indicate that t h e i r structures are highly organized and s p e c i f i c , so that nearly every one of the thousands of atoms which constitute a protein molecule occupies a d e f i n i t e p o s i t i o n . It must be concluded that polypeptide chains, no matter what t h e i r secondary structure may be, must somehow be folded to produce the compact and very nearly round mole-cules which comprise t h e i r t e r t i a r y structure. (b) Isolation The term " i s o l a t i o n " applied to proteins implies the obtaining of preparations with high s p e c i f i c a c t i v i t y per quantitative, unit. Schwimmer and Pardee (1953) mentioned the object of protein " i s o l a t i o n " i s to remove as completely as possible a l l proteins except that p a r t i c u l a r component in which resides the desired a c t i v i t y . Neilands and Stumpf (1958) suggest that " i s o l a t i o n " should be reserved for those rare instances where an investigator has t r i e d and f a i l e d to demonstrate the chemical contamination of his preparation. The term " i s o l a t i o n " used here implies the preparation of soluble concentrated proteins, which does not necessarily indicate that i t contains a single component. Isolation methods s i g n i f i c a n t l y influence the f i n a l r e s u l t s as the i n t e g r i t y of the b i o l o g i c a l product can be maintained only with a consistant y i e l d of that product under optimal conditions. (4) As there i s detailed information available regarding i s o l a t i o n methods i n the l i t e r a t u r e , t h i s problem w i l l be dealt with b r i e f l y i n t h i s report. The choice of source in the protein i s o l a t i o n i s very important. Sources d i f f e r widely i n the amounts of protein they contain; and also one source may contain undesirable material which i s absent i n the other. Also the source should be available in such amounts that a large quantity of the protein i s obtained. In the case of bacteria, the c e l l s are grown under optimal conditions fo r the production of the desired protein. The protein from the source i s either extracted by a solvent or precipitated by "common ion e f f e c t " . A number of i s o l a t i o n methods are used: 1) Sedimentation, 2) Extraction, 3) Salt Fractionation, and 4) Solvent Fractionation. (c) Protein S o l u b i l i t y and P r e c i p i t a t i o n Proteins are soluble in pH's away from t h e i r i s o -e l e c t r i c points but denaturation tends to occur. The presence of neutral s a l t s at high concentration, absence of water, pH close to i s o - e l e c t r i c point, processing and storage at low temperatures, the presence of l i p i d s (in the case of l i p o -proteins) , carbohydrates (especially in the case of muco-proteins), metal ions (metallo-proteins and metal-activated enzymes), enzyme substrates for preserving enzymes, toluene, etc., decrease the denaturation of proteins. Proteins can be obtained in the soluble form with the lea s t denaturation at a pH away from the i s o - e l e c t r i c point in the presence of (5) adequate amounts of neutral s a l t s . The same protein can be p r e c i p i t a t e d near the i s o - e l e c t r i c point. Temperature, pH and the e l e c t r o l y t e in which the protein i s suspended w i l l determine the s o l u b i l i t y of that protein. Denaturation, p r e c i p i t a t i o n and s o l u b i l i t y of proteins are dealt with in d e t a i l by Alexander & Block (1960) and Fox and Foster (1957). (°0 Protein P u r i f i c a t i o n Separating a single protein species which s a t i s f i e s the prevalent c r i t e r i a of purity i s c a l l e d protein p u r i f i c a -t i o n . There are a number of chemical methods used i n protein p u r i f i c a t i o n which w i l l not be elaborated on here, e.g., f r a c t i o n a t i o n by organic solvents l i k e ethanol, ether, acetone, methanol, dioxane and others; and the use of heavy metal ions l i k e Zn, Pb, Hg, Fe, Cu, etc. Processes important in research include fractionations by acid and s a l t s . Care must be taken in acid p r e c i p i t a t i o n s to provide enough neutral s a l t s before p r e c i p i t a t i o n to cut down denaturation. P r e c i p i t a t i o n by neutral s a l t s i s the safest way of f r a c t i o n a t i o n . 1. Repeated P r e c i p i t a t i o n . The most commonly used s a l t f o r p r e c i p i t a t i o n i s (NH ) SC> . It i s soluble i n aqueous 4 2 4 solution and consequently i s very e f f e c t i v e as a p r e c i p i t a t i n g agent. Sodium sulphate has also been used c h i e f l y because i t does not introduce nitrogen into the protein samples. Repeated p r e c i p i t a t i o n with (NH ) SO. at various l e v e l s of concentration 4 2 4 may y i e l d a c e r t a i n l e v e l of p u r i t y . Alexander and Block (1960) (6) describe a modification of the older methods o f - s a l t i n g out proteins which i s d i f f e r e n t i a l s a l t i n g i n . The proteins are pre c i p i t a t e d as a mixture and the p r e c i p i t a t e i s extracted by s a l t solutions of gradually decreasing strength. They also mention the modification of the " s a l t i n g out" method by Cohn. Instead of adding the s a l t solution d i r e c t l y to the protein sample, the protein sample i s put into a d i a l y s i n g sac which i n turn i s placed i n the s a l t s olution. The advantage of t h i s method i s that sudden changes i n the com-posi t i o n of the protein solution are avoided, with a con-sequent reduction of the co - p r e c i p i t a t i o n of protein f r a c t i o n s . 2. Adsorption Chromatography. This method includes two d i s t i n c t processes: a) adsorbent isput i n the sample of protein to which the protein adsorbs and i s then extracted by either simply the addition of a buffer or by d i a l y s i s of the adsorbed material against the required buffer. This method has been successful for quite a long time. Common adsorbents used are Alumina-gel, Benzoic Acid, Calcium Phosphate gel, hydroxides, sulphides and c e l l u l o s e s , etc. b) When adsorbent i s packed into a column the procedure i s c a l l e d adsorption column chromato-graphy. After packing, the protein sample i s applied to the column and then the adsorbed protein i s eluted by stepwise elut i o n gradient. T i s e l i u s et a l (1956) describe i n d e t a i l the preparation of chromatographic columns and introduced calcium hydroxylapatite as an adsorbent. Other adsorbents commonly used are: Hydrated Ca„ (PO ) (Swingle and T i s e l i u s , ( 7 ) 1951) S i l i c a gel, (Shepard and T i s e l i u s , 1949), Diatomaceous earths, (Martin and Porter, 1951, and Porter and Press 1957) alumina (Zechmeister et a l , 1938)and starch (Simonart and Chow, 1951). These methods have been quite successful i n the separation of various proteins but there i s no single reference available where the workers claimed that t h e i r protein preparation was pure. Care must be taken i n choosing the molar concentration, pH and temperature of the eluting buffer. Strongly adsorbed proteins may require buffers of ionic strengths which in turn cause th e i r denaturation. 3. Ion Exchange Column Chromatography. The chromatographic column i s packed with ion exchange materials which are a v a i l -able in a number of d i s t i n c t v a r i e t i e s . Proteins being macro-molecular in s i z e , require a large amount of surface area for e f f e c t i v e adsorption. The mesh size of the material i s very important. There are two types of ion exchange materials a v a i l -able: resins and c e l l u l o s e s : Resins: IRC-50 (XE 64) - Hirs et a l (1953) 200-400 mesh. Amberlite IRC-50 - Huisman & Prins (1957). Dowex 50 (NH 4) + - Sober et a l 1949 200-400 mesh Dowex 2 (Cl~) - Bowman (1955) 200-400 mesh. Amberlite IR-4B (Acetate") Waley (1957) A Cation Exchange Resin . on C e l i t e F eitelson & Partridge (1956) (8) Celluloses CM-Cellulose Peterson & Sober (1956) Cation exchanger. DEAE Cel l u l o s e Peterson & Sober (1956) Anion exchanger. Ecteola Cellulose Peterson & Sober (1956). Sulphoraethyl Cellulose Porath (1957). TEAE Cellulose Porath (1957). The preparation of the above resins and c e l l u l o s e s has been reviewed by Alexander and Block (1960) and f o r detailed information, reference may be made to the individual authors. The grain s i z e of the r e s i n , technique of packing the column, ioni c strength and pH of the buffers and the temperature are the important factors which influence the ion exchange chroma-tography of proteins. The mechanism of ion exchange involved in the case of the chromatography of amino acids i s well under-stood. Proteins on the other hand are large molecules and i t i s d i f f i c u l t to v i s u a l i s e the mechanism. Polyvalency of proteins influences the separation as does the i s o - e l e c t r i c point, the dimensions and molecular weight of the protein. Cytochrome-C with an i s o - e l e c t r i c point around pH 9.1 (Hirs et a l 1953) was p u r i f i e d on IRC-50 while proteins with i s o - e l e c t r i c points on the a c i d i c side may not y i e l d the same r e s u l t s . It i s also a well known f a c t that proteins, with t h e i r i s o - e l e c t r i c points on the basic side are more e a s i l y separated than those with i s o - e l e c t r i c points on the a c i d i c side. Boardman and Partridge (1955) noted that the physical f a c t o r s involved i n the bonding (9) of proteins at low pH's are hydrogen bonds and the Wander-Waal's forces, most probably due to removal of a layer of e l e c t r o -s t a t i c a l l y bound water. Detailed physical c h a r a c t e r i s t i c s and chromatographic behavior of proteins i s reviewed by Turba (1960) and Zittle,(1953). Ion exchange c e l l u l o s e s have been quite useful i n separating various serum proteins. (Sober & Peterson 1956, 1961) , and various other proteins — (Zimmerman et a l , 1962, Aposhr.ian.. and Romberg, 1962, Garen et a l 1960, Shukuya 1960, Maxwell 1960, Snoswell 1959, Crawford and Yanofsky 1959, Levin 1958 and Malmstrom 1957). The references given here deal with p u r i f i c a t i o n of proteins of b a c t e r i a l o r i g i n and some viruses. Our experience i n t h i s laboratory has shown that DEAE c e l l u l o s e (Selectacel) has been the best and most convenient ion exchange material f o r our purposes. The toxic proteins produced i n t h i s laboratory were not even adsorbed on Dowex-50 while polysterene showed only p a r t i a l adsorption. It i s sometimes advisable to use more than one ion exchange material i n a stepwise manner. There i s evidence i n the l i t e r a t u r e that t h i s approach has been successful i n several instances (Zimmerman et a l , 1962, Aposhrian Kornberg 1962) . Materials used for the p u r i f i c a t i o n of proteins of b a c t e r i a l o r i g i n other than c e l l u l o s e are as follows: (10) Amberlite IRC-50 — Brunish & Mozersky. XE-67 — Shainoff (1956). Amberlite XE-64 — Zimmerman (1962). Neilands and Stumpf (1958) believe that ion exchange chromatography i s e s s e n t i a l l y an electrophoretic separation in which the r e s i n serves as one electrode and gravity as the other. It may yet develop into a valuable tool as chemical industry places more new resins i n the hands of protein chemists (Colowick & Kaplan 1955). 4. Zone Electrophoresis . A large number of communications have appeared during recent years dealing with the e l e c t r o -migration of proteins, peptides and amino-acids i n a s t a b i l i z i n g media. If the s t a b i l i z i n g medium used i s paper, the technique i s c a l l e d paper zone electrophoresis. The major parts of a simple, low voltage, electrophoresis apparatus are tv/o electrodes, one p o s i t i v e and one negative; a f i l t e r paper which is dipped into the buffer present i n buffer troughs b u i l t on either side and a glass cover to cut down the evaporation of buffer. Levels of buffer on each side are the same. The sample i s applied, with great care not to scratch the paper, with a smooth-tipped c a p i l l a r y or c a p i l l a r y tube. The l i n e of application must be at least 7-8 mm. clear of the both edges. Concentration of the protein sample should be 2-10% and must not exceed 0.005t>ml/cm. The choice of buffer i s dependent on the i s o - e l e c t r i c point of the protein sample to be separated. A p o t e n t i a l of 5-10V/cm i s applied across the s t r i p ; high (11) po t e n t i a l s generate more heat and reproducible r e s u l t s are d i f f i c u l t to obtain. A known protein sample should be tested before applying the unknown sample. The proteins are detected by staining after heating the s t r i p at 110° for 20 minutes. Some of the available dyes used are Amido-Black 10B, lissamine green, ninhydrin and bromophenol blue. Zones can be marked on the s t r i p by the absorption.in u l t r a - v i o l e t l i g h t p r i o r to staining and the zones can be cut and eluted for quantitative purposes. The main physical f a c t o r s a f f e c t i n g migration rates on paper are the type and concentration of buffer, control of evaporation, electro-endosmosis, and the e l e c t r i c f i e l d . The application of paper zone electrophoresis i s limited and cannot be successfully used in quantitative research. This technique i s a valuable t o o l i n the i n i t i a l q u a l i t a t i v e work. Zone electrophoresis in s t a b i l i z i n g media such as granular starch, powdered c e l l u l o s e or r e s i n beads o f f e r s con-siderable advantages over paper zone electrophoresis,which are: 1. Large amounts of proteins can be separated during one run and are available f o r further a n a l y t i c a l work. 2. The evaporation problem i s not as acute, and 3 . Some of the media are suitable f o r the determination of m o b i l i t i e s and i s o - e l e c t r i c points. The apparatus used i n most laboratories i s the same as that o r i g i n a l l y devised by (12) Kunkel and T i s e l i u s (1951) f o r paper zone electrophoresis. A s i g n i f i c a n t development was made by Smithies i n 1955 with the introduction of starch gel as a s t a b i l i z i n g media in zone electrophoresis. A combination of electrophoresis in agar gel with s e r o l o g i c a l p r e c i p i t a t i o n c a l l e d Immuno-electrophoresis was developed by Graham and Williams (1955) in which, afte r a normal electrophoretic run anti-serum i s applied lengthwise on one side. The antiserum diffuses through the s t a b i l i z i n g media and gives a s p e c i f i c p r e c i p i t a -tion reaction. Separations are also possible on a micro-scale using c e l l u l o s e acetate (Kohn, 1957) which i s an ideal medium for immuno-electrophoresis (Consden & Kohn, 1959). Zone electrophoresis in v e r t i c a l columns has been tech n i c a l l y developed by Haglund and T i s e l i u s (1950), Porath (1954), and Flodin and Kupke (1956), the obvious advantage being the c o l l e c t i o n of fra c t i o n s as i n Column chromotography. For the detailed study of these techniques, reference must be made to the o r i g i n a l authors and valuable information i s also given by Bailey (1962). There are few references available where these techniques are exploited f o r the p u r i f i c a t i o n of proteins of b a c t e r i a l o r i g i n . The s p e c i f i c a c t i v i t y of a protein/unit can be determined either with the help of u l t r a v i o l e t absorption at the suitable wave length or by c a l c u l a t i n g the amount of nitrogen/unit protein. The nitrogen content of a protein can best be obtained from samples free of carbohydrates and (13) l i p i d s using Micro-Kjeldahl method (Bailey 1962). (E) C r i t e r i a of Purity The c h a r a c t e r i s t i c of a pure protein i s that i t gives one boundary under u l t r a c e n t r i f u g a t i o n and electropho-r e s i s . These c r i t e r i a of purity are a "must" i n contemporary techniques apart from other determinant c h a r a c t e r i s t i c s . (1) C r y s t a l l i z a t i o n : This was considered to be a c r i t e r i o n of purity in the late 1920's . and reports were made even more recently where (Lamanna, 1946) emphasis.was l a i d on c r y s t a l l i z a t i o n . However, several c r y s t a l l i n e proteins have been shown to be quite impure; these include Type "A" botulinus toxin (Wagman & Bateman 1951, Wagman, 1954), heart l a c t i c dehydro-genase, ribonuclease, lysozyme and Sumner's urease (c.f. Neilands and Sturapf 1958). Therefore c r y s t a l l i n i t y i n i t s e l f i s an extremely unreliable index of p u r i t y . Also c r y s t a l s formed under c e r t a i n conditions may d i f f e r e n t i r e l y i n configuration from those formed under another set of con-d i t i o n s . Isomorphous proteins may be present in the same c r y s t a l s , i n d i c a t i n g f a l s e v i s u a l purity (Fox and Foster 1957). Baronwski (c.f. Schwimmer and Pardee, 1953) has shown that c r y s t a l l i n i t y i s c e r t a i n l y an i n d i c a t i o n of a c e r t a i n l e v e l of p u r i t y and c r y s t a l l i n e proteins are usually not denatured. (2) B i o l o g i c a l c r i t e r i a . Determination of the s p e c i f i c b i o l o g i c a l a c t i v i t y per unit may be used as one of the c r i t e r i a of purity. Pure proteins show much higher b i o l o g i c a l a c t i v i t y per unit than crude preparations. (14) (3) The chemical structure of a preparation must be consis-tent at a l l times with the e x i s t i n g properties of the pure component. (4) P u r i f i e d protein w i l l in most cases give a single peak under ion-exchange or adsorption column chromotography. Changes i n pH, io n i c strength as well as the buffer are e s s e n t i a l l y t r i e d i n order to confirm the purity of the sample. (5) Paper, starch and immuno-zone electrophoreses ' techni-ques indicate a very high l e v e l of purity and one or a l l must be t r i e d depending on the sample. The choice of buffers i s very important i n these techniques. (6) U l t r a c e n t r i f u g a l analysis. Protein preparations are analysed with the ultracentrifuge f o r establishing purity and fo r c a l c u l a t i n g the sedimentation c o e f f i c i e n t . This technique was developed i n 1922 when Svedverg started exploring the f i e l d . Beckman-Spinco i s the supplier of a l l ultracentrifuges in North America. Ultracentrifugation includes two major techniques; the sedimentation v e l o c i t y and the sedimentation equilibrium method. The former method i s used most commonly for the determination of purity, while with the l a t t e r method the d i f f u s i o n c o e f f i c i e n t can be calculated. The sedimentation v e l o c i t y method i s based on spin-ning a protein preparation at a very high speed i n an u l t r a -centrifuge. During t h i s process, the heavier solutes leave the solvent and accumulate towards the periphery of the c e l l . (15) This migration of the solute p a r t i c l e s leaves a region i n the c e l l containing only solvent p a r t i c l e s and a zone where the concentration of the solute i s uniform. There i s also a t h i r d place i n addition to the above where the concentration varies with distance from the axis of rotation. This i s c a l l e d the boundary. The sedimentation v e l o c i t y method, i n b r i e f , i s based on observations, by o p t i c a l methods, of'the movement of the boundary which in turn i s a measure of the movement of the solute molecules in the plateau region. The rate at which the solute p a r t i c l e s move i s a function both of the. molecular weight of the solute and of the functional resistance which other molecules or p a r t i c l e s experience as they move through the solvent. Purity of the preparation can be indicated i f one uniform boundary i s shown after an adequate length of time. Faulty observations may be made i f the preparation used was of high concentration and no analysis was made at d i l u t e l e v e l s . This method i s not the ultimate technique in the determination of purity as a single boundary may be shown with d i f f e r e n t proteins having the same sedimenta-tion c o e f f i c i e n t ; a detailed review has been given by Schachman (1957). (7) D i f f u s i o n Electrophoresis. D i f f u s i o n i s the movement of one solute into another when the l a t t e r i s of lower con-centration than the former. The d i f f u s i o n c o e f f i c i e n t i s the rate of d i f f u s i o n from one solute to the other i n a given period of time under a dr i v i n g force. D i f f u s i o n electropho-r e s i s i s an apparatus by which the d i f f u s i o n c o e f f i c i e n t of a protein i s determined. Molecular weight can then be calculated (16) from the sedimentation studies and d i f f u s i o n c o e f f i c i e n t . The T i s e l i u s electrophoresis c e l l , with only s l i g h t modifica-tions, i s the id e a l c e l l . Schachman (1957) has covered the working of t h i s c e l l and i t s modifications, etc. wiith great d e t a i l arid l u c i d i t y . The r e s u l t s of d i f f u s i o n electrophoresis are also used as a c r i t e r i o n of purity. Calculations for molecular weight determinations as well as other d i f f u s i o n techniques have been reviewed by Alexander and Block (1961). The T i s e l i u s electrophoresis c e l l i s most frequently used for determining homogeneity and electrophoretic mobility of protein preparations. The material i s applied to the c e l l and subjected to an e l e c t r i c current. The d i f f e r e n t components form concentration gradients under these conditions which can be observed with Schlieren optics. Different components separate according to t h e i r m o b i l i t i e s under the conditions of the run. In the case of a homogeneous preparation only one moving peak w i l l be observed. F. Amino Acid Analysis Preparations with:. 13.5% or more nitrogen content are considered as pure protein preparations and amino acid analysis may be c a r r i e d on these preparations (Alexander and Block 1961 Vol. 2) which i n no case means that there i s only one protein species.present.v.'Amino.;-icid, analysis of preparations containing more than one component i s a waste of time. (17) The f i r s t step in the amino-aeid analysis i s the hydrolysis of proteins. It i s brought about with addition of acids such as hydrochloric, sulphuric, hydriodic, formic and others; the detailed procedure f o r these acids i s given by Alexander & Block (1961, Vol.2). Column chromatography of the hydrolysed protein by the methods of Moore & Stein (1954) w i l l give the researcher both q u a l i t a t i v e and quantitative r e s u l t s regarding the amino.-acid constituents and r a t i o s for the protein being studied (Tristram, 1963). Under these conditions, an empirical formula f o r the p u r i f i e d protein may be calculated. (18) CHAPTER II  Review Literature on Botulinus Proteins "There i s an old Dutch word for chemistry, 'scheikunde', which l i t e r a l l y means the 'art of separation*. Indeed, separation methods form the basis of chemistry, and the d e f i n i t i o n of a pure chemical substance ultimately depends on separative operations". ( T i s e l i u s 1961; c f . Heftman 1961). "Scheikunde" or the "art of separation" also forms the basis fo r biochemical research. During the past two decades, there has been consider-able interest in the problems associated with determining the s p e c i f i c a c t i v i t y and chemical structure of ce r t a i n deadly proteins of b a c t e r i a l o r i g i n , and e f f o r t s have been made to obtain these toxins i n the pure state. One group of such substances are the exotoxins produced by types, A, B, C (alpha and beta), D, and E, of Clostridium botulinum. The toxins, for the experimental work, were either obtained from f i l t r a t e s or extracted from whole b a c t e r i a l c e l l s . There were d i f f e r e n t methods used f o r the production and p u r i f i c a t i o n of various types of botulinus toxins which are discussed in d e t a i l as follows: (a) Clostridium Botulinum Type A The p u r i f i c a t i o n of t h i s toxin was attempted as fa r back as 1906 but s i g n i f i c a n t contributions v/ere not made u n t i l 1926 when Sommer et a l used c o l l o i d a l A l (0H)„, (19) to which the toxin i s strongly adsorbed. They t r i e d to elute the adsorbed toxin with weak acetic acid (molarity not specified) at pH 3.8 and ammonia (pH 8.4). This was unsuc-ce s s f u l but t h i r t y to f i f t y percent of the adsorbed toxin was f i n a l l y recovered after treatment with a 0.5% solution of (NH ) HPO + 0.1% glycerol at 28° C for f i v e hours. The 4 2 4 toxin was freed of s a l t s by d i a l y s i s and evaporated to dry-ness at 40° C. This dried toxic preparation was soluble in water. A drop i n t o x i c i t y was observed during d i a l y s i s and the f i n a l y i e l d was 0.15 to 1.8%. Sommer and Snipe (1928) investigated the adsorp-t i v e properties of type A toxin further on c o l l o i d a l alumina, , and elucidated the role of the pH factor in t h i s process. The toxic product obtained gave a pos i t i v e Biuret test, a s l i g h t Molischtest and contained a small amount of gelatihase. These workers (Snipe and Sommer 1928) l a t e r showed that the toxin was precipitated by lowering the pH of the toxic f i l -t r a t e . The optimum pH for p r e c i p i t a t i o n was found to be 3.0 and gave a y i e l d of 28.5%. Further attempts to purify the toxin by repeated d i s s o l u t i o n at pH 7.0 and re p r e c i p i t a t i o n at pH 4.4 resulted in the loss of most of the toxic a c t i v i t y . Sommer et a l (1928) attributed the loss of t o x i c i t y during d i a l y s i s to a very small extent to d i f f u s i o n . They noted that a large amount was i r r e v e r s i b l y inactivated,for which no explanation was given. The maximum s t a b i l i t y of (20) the toxin was also reported t o be between pH 4.0 and pH 5.0. Sommer (1936) obtained preparations of high t o x i c i t y by using repeated acid p r e c i p i t a t i o n s with N/10 HC1 between pH 3.5 and 4.0 and with subsequent di s s o l u t i o n i n sodium acetate buffer (molarity not s p e c i f i e d ) . The prepara-tions contained l e t h a l doses of 4.10 - 9 gm = 2.10 -7 gm per Kg mouse. The y i e l d of the f i r s t p r e c i p i t a t e was 50% but decreased after further r e p r e c i p i t a t i o n s . He also noted that the toxin was gradually destroyed at room temperature and that the presence of t r i v a l e n t ions enhances t h i s e f f e c t . Stockinger and Ackerman (1941) found that toxic f i l t r a t e s were stable between pH 1.5 and pH-8.5 and c a r r i e d out f r a c t i o n a l p r e c i p i t a t i o n s of toxic f i l t r a t e s at pH 4.0 and 1.5. They found that the bulk of the toxin was precipitated at the .Mter pH. The concentrated material was treated with "takadiastase" to remove carbohydrate-and phosphate-containing material (amounting to 10 - 20%), and preliminary analyses showed that the p u r i f i e d toxin i s a simple, unconjugated protein of the albumin c l a s s and contained l e s s than 1 percent nitrogen and four percent s u l f u r . It contained 10,000 to 20,000 MLD/mg toxin. They also noted the significance of d i s u l f i d e bonds and observed that the destruction of these bonds brought about the loss of t o x i c i t y . Abrams and co-workers (1946) reported that type A toxin was stable at room temperature between pH 1.0 and 6.0 (the maximum being between pH 4.0 - 5.0) but that the toxin was rapidly destroyed at pH's above 7.0. They used (21) Sommer*s (1936) acid p r e c i p i t a t i o n technique and adjusted the toxin f i l t r a t e s to pH 3.5. The preci p i t a t e d toxin then was t h r i c e extracted i n 1/4 the o r i g i n a l volume of 1% sodium acetate solution at pH 6.5; and the three extractions were pooled and the insoluble material discarded. The toxin was precipitated again at pH 3.5; washed twice in d i s t i l l e d water; redissolved in 1/4 the o r i g i n a l volume of 1% sodium acetate solution, then prec i p i t a t e d with 10, 20, 40 and 50 per cent ethanol; 'ftn& further p r e c i p i t a t e d with saturated Nag SO^ at pH 6.5 at room temperature. The material obtained was soluble i n d i s t i l l e d water and f r a c t i o n a l p r e c i p i t a t i o n at 0.18 and 0.4 saturated Nag SO^ was ca r r i e d ©ut. The toxic f r a c t i o n was contained i n the 0.4 saturated Na 2 SO^ preparation, had a t o x i c i t y of 60 x 10 mouse LD/mg N., and had an i s o -e l e c t r i c point at pH 5.6. This toxin gave a pos i t i v e test f o r carbohydrates and phosphorus. The toxin was electr©-ph o r e t i c a l l y homogeneous. The authors note that the "homogeneous" f r a c t i o n had fewer mouse LD/mg N than did ce r t a i n impure f r a c t i o n s . Another toxic f r a c t i o n with t o x i c i t y of 220 x 10 MLD/mg N was obtained by using a s l i g h t l y d i f f e r e n t procedure. The buffer used i n t h i s case was 1% sodium phosphate buffer at pH 6.8. The rest of the procedure was es s e n t i a l l y the same as given before and the recovery rate was 34 per cent. This component was twice c r y s t a l l i z e d i n 0.10 to 0.30 saturated CM1 4) 2S0 4 at 4° C. (22) This t©xic f r a c t i o n did not s a t i s f y the c r i t e r i a of purity used by Abrams et a l which were: 1) maximum t o x i c i t y per mg nitrogen; 2) electrophoretic homogeneity and 3) a single u l t r a v i o l e t absorption band at 278W^X. The procedure for p u r i f i c a t i o n developed by Lamanna et a l (1946) varied to a cer t a i n extent from the technique used by Abrams et a l (1946). The toxin was p r e c i p i -tated at pH 3.5 with 2 N HC1, the supernatant siphoned o f f , and the pr e c i p i t a t e washed i n d i s t i l l e d water. The washed pr e c i p i t a t e was resuspended i n water, t© which 1 M NaCl and 0.075 M sodium acetate solutions were added and the pH adjusted t© 6.5. The pr e c i p i t a t e was discarded and 1/6 to 1/10 volume C P . chloroform was added and shaken under C0 2 f o r f i v e minutes at pH 5.8 and 6.1. The gel and the excess chloroform were discarded and the toxin present i n the aqueous gel was precipitated with 20% saturated (NH 4) 2 S0 4 # The pr e c i p i t a t e was obtained by lowering the pH to 5.0 by the addition ©f HC1. The toxic p r e c i p i t a t e was resuspended i n 1 M NaCl + 0.075 M sodium acetate solution at pH 6.5. To t h i s suspension, 0.9 gm (NH 4) 2S© 4 / m i toxin was added and a coloured p r e c i p i t a t e formed which was discarded. The super-natant was allowed t© stand at 4® C overnightj the toxic c r y s t a l s which formed had a nitrogen content of 14.3%. The 9 • y i e l d was 15 t© 20% and the t o x i c i t y was 4.5 x 10. ~ mouse LD 50/ mg N. The pure toxin had the properties of a protein and fourteen amino acids were i d e n t i f i e d as well as phosphate and su l f u r . (23 ) Kegeles ( 1 9 4 6 ) calculated the d i f f u s i o n constant for the c r y s t a l l i n e toxin (Abrams et a l 1946 and Lamanna et a l 1946) and recorded the v i s c o s i t y measurements. The molecular weight of the toxin was reported to be 1 , 1 3 0 , 0 0 0 . (N.B. Inhomogeneity was observed e l e c t r o p h o r e t i c a l l y a f t e r these calculations«). Putnam et a l ( 1 9 4 6 ) studied the electrophoretic, sedimentation and d i f f u s i o n c h a r a c t e r i s t i c s of Lamanna's ( 1 9 4 6 ) c r y s t a l l i n e toxin i n 0 . 1 M sodium acetate buffer at pH 4:; 3 8 . They noted that the boundary spread was greater than that attributable to d i f f u s i o n alone. The molecular weight of the material sedimenting with a c o e f f i c i e n t of S 2 Q W - 1 7 . 3 0 was calculated as 9 0 0 , 0 0 0 . The molecules were assumed to be e l l i p s o i d s and an estimate that there were 2 . 1 x 10^ molecules per Mouse LD was made. 50 Buehler et a l ( 1 9 4 7 ) studied the elemental and amino acid composition of Lamanna et a l ( 1 9 4 6 ) toxin and i d e n t i f i e d nineteen amino acids. The minimum possible molecular weight was reported to be 4 4 , 9 4 4 and an empirical formula was given. The presence of phosphorus was f e l t to be an impurity (possibly the residue of nucleo-proteins). The c r i t e r i a of pur i t y used were ul t r a c e n t r i f u g a t i o n , electrophoretic and sero l o g i c a l behavior. Putnam and h i s associates used the chloroform shaking method i n 1 9 4 8 to pu r i f y the toxic material. They obtained photographs of the electrophoretic migration of the ( 2 4 ) c r y s t a l l i n e toxin and calculated the d i f f u s i o n c o e f f i c i e n t . The sedimentation c o e f f i c i e n t was reported to be S nW-17.35 and the molecular weight 900,000. Boundary spreading indicated the polydisperse character of the toxin molecule. The immuno-l o g i c a l homogeneity was also studied and indicated. Lamanna and Doak (1948) gave a f u l l report on the se r o l o g i c a l behavior of Lamanna's (Lamanna et a l 1946) c r y s t a l l i n e toxin. They claimed that the antitoxin response in Rabbit and Horse v/as complete and that the toxin was a single antigenic component. Ca r l Lamanna (1948) worked on the hemagglutination property of the c r y s t a l l i n e toxin. The amorphous as well as the c r y s t a l l i n e toxins reacted with the red blood c e l l s of chickens, guinea-pigs, rabbits, sheep, and man, although the toxin i t s e l f was not adsorbed onto the red blood c e l l s . The a c t i v i t y of hemagglutination and t o x i c i t y was decreased by treatment with formaldehyde. Lamanna and Lowenthal (1951) further studied the haemagglutinating and the toxic a c t i v i t i e s and a material was separated which caused the hemagglutinating a c t i v i t y . The type s p e c i f i c antiserum neutralized both the a c t i v i t i e s while type non-specific antiserum did not. The toxic a c t i v i t y was, however, more sensitive to heat while the hemagglutinating a c t i v i t y was sens i t i v e t© an acid pH and was neutralized by type B antitoxin. Oudin-plate technique (25) also demonstrated the se p a r a b i l i t y of the two properties when type A antitoxin was used. The multiplicity of type A c r y s t a l l i n e toxin c o n f l i c t e d with the apparent homogeneity of material shown by ul t r a c e n t r i f u g a t i o n and electrophoresis. In the l i g h t of t h i s l a t e r data, they hypothesized the formation of a stable complex of the two substances with the c r y s t a l l i z a t i o n technique applied. This was l a t e r confirmed by Lowenthal and Lamanna (1953). Lamanna and Aragon (1956) reported the i s o l a t i o n of a protein component from the c r y s t a l l i n e toxin which was responsible for the hemagglutinating a c t i v i t y . The p e c u l i a r i t y of the high molecular weight of type A c r y s t a l l i n e toxin was noted by Wagman and Bateman (1951). They showed that the sedimentation constants varied with the concentration of the preparation used, and that by r a i s i n g the pH, d i s s o c i a t i o n of the large molecules took place and a slowly sedimenting component having considerable toxic a c t i v i t y with no haemagglutinating a c t i v i t y could be iso l a t e d . Wagman in 1954 reported the sedimentation c o e f f i -c i e n t of t h i s active component to be S 2 0 ^ = 6.55 and the mole-cular weight 70,000. He kept the toxin i n 0.05 M acetate buffer of pH 3.8 at 5°C. He prepared the c r y s t a l l i n e toxin by using Abrams (1946) method and estimated the i s o - e l e c t r i c point to be pH 5.5. The c r y s t a l l i n e toxin was dialyzed at pH 6.5 to 8.0 with phosphate buffers of ionic strengths 0.13 and 0.5. (higher ionic strength buffers gave better separation). (26) With t h i s treatment a product was obtained which had a sedimentation c o e f f i c i e n t of S2o w = 6.55 and contained f i f t e e n percent of the t o t a l toxic a c t i v i t y . This component poly-merised at pH 3.8 and a heavy component was formed which was obtained with phosphate buffer of ioni c strength 1.0 (Wagman 1954). The low molecular weight material had two to three times the s p e c i f i c a c t i v i t y of the large complex. The heavy component was si m i l a r to the o r i g i n a l component and he considered the p o s s i b i l i t y that toxin molecules were aggre-gated with non-toxic material. H a l l i w e l l (1954) extracted the toxin from lysed c e l l s and pre c i p i t a t e d i t i n acid c i t r a t e buffer containing 0.18 M sodium c i t r a t e (Hg) and 0.02 M sodium c i t r a t e (Na2) at pH 4.0. Further p r e c i p i t a t i o n was done with ammonium sulphate. The samples contained 70 x lO^rMLD/mg N. However, further batches were prepared by t h i s method with t o x i c i t i e s comparable to those obtained by Lamanna et a l (1946). Both the toxins were c r y s t a l l i n e and homogeneous el e c t r o p h o r e t i c a l l y . The two preparations d i f f e r e d greatly i n s t a b i l i t y ; the low a c t i v i t y material being r e a d i l y i n a c t i v i a t e d in 2 minutes in 3.5 N HC1 at 16.2°C. Boththe toxins were stable i n buffers of pH 5.0 to 6.5 at 0° C. The low t i t r e material was rapidly destroyed at 30° C at pH 7.0 while the high t i t r e material was stable under these conditions f o r 26 hours. (27 ) A high state of purity was obtained by Duff et a l ( 1 9 5 2 , 1 9 5 7 ) by acid p r e c i p i t a t i o n at pH 3 . 5 . The pr e c i p i t a t e was washed with d i s t i l l e d water and the toxic component was extracted from the p r e c i p i t a t e with 0 . 0 7 5 M CaClg and reprecipitated at pH 3 . 7 . The pr e c i p i t a t e was again dissolved i n phosphate buffer at pH 6 . 8 . Further p r e c i p i t a t i o n was done with f i f t e e n percent ethanol in the cold. This preparation was c r y s t a l l i z e d by p r e c i p i t a t i o n with (NH4)2 SO4 and had a s p e c i f i c a c t i v i t y of 6 / 269 x 10 mouse intraperitoneal L D ^ Q /mg N . This toxin appeared to be homogeneous i n the a n a l y t i c a l u l t r a c e n t r i f u g e . Since 1 9 5 7 , no further data have come to l i g h t regar-ding the p u r i f i c a t i o n and characterization of botulinus type A toxin. The accumulated information documented above strongly indicates that the c r i t e r i a f o r purity at our disposal today have not been s a t i s f i e d regarding t h i s material. The techniques employed by most of the workers involved in t h i s problem were based on the i n i t i a l p r e c i p i t a -t i o n of toxic matter with subsequent repeated p r e c i p i t a t i o n s obtained either by lowering the pH or " s a l t i n g out". A s i g n i f i c a n t departure from these methods was introduced by Lamanna ( 1 9 4 6 ) with h i s "chloroform shaking process", the advantage of which i s doubtful. An unduly high significance has been placed on the obtaining of the toxic component i n a c r y s t a l l i n e form. C r y s t a l l i z a t i o n of material indicates merely that one species of c r y s t a l has formed in a given preparation and i t i s abundantly c l e a r that two or more d i s t i n c t molecular (28) structures may be involved i n the formation of such a c r y s t a l (Neilands and Stumpf, 1958). The i n i t i a l biophysical studies c a r r i e d out on c r y s t a l l i n e type A toxin indicated imhomogeneity, both i n the ultracentrifuge (Putnam et a l 1946), wherein i t was noted that the boundary spread was greater than could be attributed to ordin ary d i f f u s i o n , and during electrophoresis (Kegeles, et a l , 1946). Later data (Wagman and Bateman 1951, 1953, and Wagman, 1954) showed that the c r y s t a l l i n e material could be dissociated and a smaller toxic e n t i t y i s o l a t e d . Lamanna himself (Lowenthal & Lamanna 1951) l a t e r postulated the formation ©f a stable complex during c r y s t a l l i z a t i o n . In the l i g h t ©f general knowledge today, the assumption that a protein molecule with the remarkable bio-l o g i c a l a c t i v i t y of Type A toxin has a molecular weight of about a m i l l i o n appears u n r e a l i s t i c ; and one can deduce with some certainty that t h i s protein has not, t© date, been p u r i f i e d to a degree wherein i t s properties may be s a t i s f a c t o r i l y studied. (b) Clostridium botulinum Type B Lamanna and Glassman (1947) developed a technique for the production and p u r i f i c a t i o n of Type B toxin. The 'OKRA* s t r a i n ©f Type B was grown at 34° C for fourteen days and the culture was brought to pH 4.0 with 2 N HC1. The culture was allowed to stand at room temperature and a (29) p r e c i p i t a t e formed which gave a 90% recovery of the o r i g i n a l t o x i c i t y of the whole culture. The supernatant was d i s -carded and the p r e c i p i t a t e was suspended in 1/40 of the o r i g i n a l volume in d i s t i l l e d water. The pH was brought to 2.0 with 2 N HC1. The p r e c i p i t a t e was discarded and the f i l t r a t e contained 80% of the o r i g i n a l toxic a c t i v i t y . This c l e a r f i l t r a t e was brought to pH 4.0 with 2 N NaOH and a f l o c c u l e n t p r e c i p i t a t e was obtained (the authors noted that the recovery rate was more consistant and higher at pH 5.0) which was then resuspended i n 1/4 of the o r i g i n a l volume i n .5 M NaCl at pH 2.0 for 10 to 15 minutes. The p r e c i p i t a t e was recovered by centrifugation and resuspended i n 1/4 o r i g i n a l volume of a c i d i f i e d d i s t i l l e d water at pH 2.0. The toxin was reprecipitated by bringing the pH to between 5.0 and 5.5. The f i n a l recovery rate was 50%. The p u r i f i e d toxin was greyish yellow when s o l i d and yellow brown in solution. It gave a p o s i t i v e biuret test and was negative f o r carbohydrates. The maximum absorption was at 277>n/< and the amino nitrogen was 15.5%. The molecular weight determination was c a r r i e d out by c a l c u l a t i n g the d i f f u s i o n c o e f f i c i e n t at pH 2.0 and was reported to be 60,000 (the electrophoretic analysis showed that pH 4.5 was on the acid side of the i s o -e l e c t r i c p o i n t ) . The toxin was insoluble between pH 4.0 and 7.0 and the presence of s a l t s decreased i t s s o l u b i l i t y i n water. Electrophoresis which was c a r r i e d out between pH 1.8 and 3.8 gave a single boundary, and the s p e c i f i c t o x i c i t y was Q 5.9 x 10 mouse LD 50/mg N. L a b i l i t y of the material appeared to be increased after p u r i f i c a t i o n . (30) Wagman and Bateman ( 1 9 5 1 ) studied the behaviour of Lamanna and Glassman's p u r i f i e d ( 1 9 4 7 ) type B toxin at pH 3 . 0 i n the a n a l y t i c a l ultracentrifuge and reported the molecular weight as 5 0 0 , 0 0 0 . Theyisolated another slow moving component with sedimentation c o e f f i c i e n t S 2 Q W = 1 0 . 7 which represented about 30% of the t o t a l toxic material. Duff and co-workers ( 1 9 5 4 ) showed that the toxin could be extracted from acid p r e c i p i t a t e s with CaClg at pH 1 . 5 to 6 . 5 , the greater recovery being between pH 1 . 5 to 2 . 5 . This material was not soluble above pH 4 . 5 . Recovery of t h i s toxin was extremely low at pH 5 . 5 to pH 6 . 5 but the component obtained aft e r further p u r i f i c a t i o n was soluble between pH 5 . 5 . and 6 . 5 . P u r i f i c a t i o n was completed by pr e c i p i t a t i n g with acid and then with ethanol in the cold. M i l l o n i g i n 1956 used the "OKRA" s t r a i n and produced toxic f i l t r a t e s with the dry casein method. A pre c i p i t a t e was obtained with (NH 4) 2 S(>4 to a f i n a l saturation of 0 . 6 at pH 7 . 0 . F i n a l p u r i f i c a t i o n was obtained i n 1 M NaCl at pH 6 . 0 with 0 . 3 saturated (NH ) SO . Duff et a l ( 1 9 5 7 ) grew the toxin in trypticase yeast extract, cystine hydrochloride and glucose. The cultures contained 2 . 0 x 1 0 6 mouse intraperitoneal LD 5 Q/ml or greater. The pr e c i p i t a t e formed by a c i d i f i c a t i o n at pH 4 . 5 was washed with d i s t i l l e d water and the toxin was extracted from i t with 0 . 0 5 M calcium chloride solution at pH 6 . 0 between 3 0 ° C and 3 5 ° C. Further p r e c i p i t a t i o n was done at pH 3 . 7 and the toxin was dissolved.in 0 . 1 M phosphate (31) buffer at pH 5.5 or 0.1 M phosphate buffer at pH 6.8. This material gave a s p e c i f i c a c t i v i t y of 262 x 10 mouse LD /mg N 50 and when examined i n the a n a l y t i c a l u l t racentrifuge, two components were obtained; one main component with S W of 14.9, and a d i f f u s e l y sedimenting component with S W of 10.9 at pH 3.0. The method used by Lamanna and Glassman (1947) for the p u r i f i c a t i o n of botulinus type B toxin i s e s s e n t i a l l y based on repeated p r e c i p i t a t i o n s at various pH's. It i s indicated i n the r e s u l t s that a large amount of t o x i c i t y was l o s t a f t e r each p r e c i p i t a t i o n and subsequent suspension. Since proteins show minimum s o l u b i l i t y near the i s o e l e c t r i c pH, the f r a c t i o n a t i o n around that pH i s v a l i d (Neilands and Stumpf 1958) but denaturation may occur i f a pH beyond the i s o e l e c t r i c point i s used (Alexander and Block 1958). Also, the lack of neutral s a l t s in the solution aids in denaturation. Wagman and Bateman (1951) showed that there were two components-in the p u r i f i e d toxin of Lamanna and Glassman (1947) and were successful in separating them. Duff et a l (1954, 1957) made no s i g n i f i c a n t con-tr i b u t i o n s towards p u r i f i c a t i o n of botulinus type B toxin as: 1) they used e s s e n t i a l l y the same technique as Lamanna and Glassman (1947),and 2) S p e c i f i c t o x i c i t y / mgN was much lower than Lamanna and Glassman's (1947) p u r i f i e d toxins. (32) It i s quite c l e a r that botulinus type B toxin obtained by these workers was {inhomogeneous and s a t i s f a c t o r y tests f o r determining the purity were not c a r r i e d out. (c) Clostridium Botulinum Type C Sterne and Wentzel (1950) developed an apparatus •7 f o r t h e i r production of toxins in carboys with inserted cellophane bags i n which the organisms were grown. They used unconcentrated corn-steep liquor (3.5% of t o t a l solids) at pH 7.2 with 1% NaCl as media. The t o x i c i t y obtained was 3.0 x 10 MLD/mg.N. No further attempts were made at t h i s time to pu r i f y the product. Boroff et a l (1952)+ extracted the toxin from 48 hour c e l l s , washed three times with d i s t i l l e d water, grown on media composed of peptic digest of l i v e r and beef muscle enriched with 1% glucose, at pH 7.0, with 1 M NaCl and 0.1 M Sodium c i t r a t e solutions kept f o r 6 days in the cold. The ~ supernatant was dialyzed against d i s t i l l e d water f o r 48 hours and a white p r e c i p i t a t e appeared. Both the p r e c i p i t a t e and the supernatant were toxi c . The supernatant and the p r e c i p i t a t e were treated separately in the following ways:-The supernatant was brought to pH 6.0 with 6N HC1 and the p r e c i p i t a t e obtained was of feeble t o x i c i t y and was discarded. The pH of the supernatant was further decreased to 4.0 and a + The culture on which Boroff et a l (1952) did t h e i r work was mistakenly understood to be of type D but was l a t e r proved to be of type C. (33) toxic p r e c i p i t a t e was recovered with p a r t i a l s o l u b i l i t y at pH 2.0. The toxin was reprecipitated at pH 4.7 (pH change was brought about by the addition of 1 N NaOH). The p r e c i p i t a t e was washed i n phosphate buffer at pH 4.5 and redissolved at pH 5.7. The pH was brought to 7.2 and the material dialyzed against 0.85% s a l t solution. The t o x i c i t y of the f i n a l product was 1.4 x 10 mouse LD 5Q,/mg N. The p r e c i p i t a t e was treated with 0.1 M sodium c i t r a t e solution and 1 M NaCl. The toxic extract was dialysed f o r two days at 0° C against d i s t i l l e d water. This toxin went into solution at pH 9.0 and was completely detoxified but was insoluble between pH 4.0 and 7.0. Both the toxins were unstable at 40° C but stable at 4° C. It was noted that heat at 60° C applied for 30 minutes completely detoxified the toxins. The type C s t r a i n , i s o l a t e d by Robinson and T h e i l e r i n 1928, was grown in media composed of 4% proteose peptone, 2.0% pancreatic digest of casein (N-Z amine type B, S h e f f i e l d , Norwich, N.Y.), 0.2% yeast extract (BBL) and 1.0% glucose, by Cardella et a l i n 1958. Four l i t r e s of media in carboys were inoculated with 10% of i t s volume of a 24 hour inoculum and incubated at 33° C f o r f i v e days. The f i l t r a t e was treated with 95% ethanol to a f i n a l concentra-ti o n of 25% for 18 to 24 hours at 5° C, a f t e r which the supernatant was discarded. The p r e c i p i t a t e was d i l u t e d to 1/8„ culture volume with d i s t i l l e d water and the volume was further d i l u t e d to 1/4 culture volume with 1 M C a C l 0 (34) ( f i n a l concentration of Cacl was 0.5M). The pH was adjusted to 5.0, s t i r r e d f o r 1 hour at room temperature and was cen-trifuge d at 4° C. The pH of the supernatant was further i n -creased t© 6.0 with 1 N NaOH, and a p r e c i p i t a t e was obtained with 50% ethanol, added to a f i n a l concentration of 15% and allowed to stand at -5° C f o r 18-24 hours. The pr e c i p i t a t e was dissolved i n 0.4 M succinate buffer at pH 5.0 and the solution c l a r i f i e d by centrifugation at 4° C. These workers indicated that ethanol p r e c i p i t a t i o n from the cultures at pH 5.5 to 6.0 was complete and the recovery was much higher than the acid p r e c i p i t a t i o n method i n which recovery was low. They also t r i e d p r e c i p i t a t i o n with 40% saturated (NEL) SO at room temperature overnight. The ^ 2 4 pr e c i p i t a t e was obtained aft e r centrifugation at 4,000 RPM at 4° C and resuspended i n water to 1/4 culture volume. The rest of the procedure was the same as used with ethanol p r e c i p i t a t e s . Boroff and co-workers used the acid p r e c i p i t a t i o n method to pu r i f y type C botulinus toxin. None of the workers characterized t h i s toxin and also f a i l e d to c l a r i f y whether t h i s type belonged to or C ^  of botulinus toxins. Thus i t i s very d i f f i c u l t to correlate the r e s u l t s obtained by various workers i n t h i s f i e l d . Lack of s u f f i c i e n t neutral s a l t s , p r e c i p i t a t i o n of toxin at pH's beyond the i s e - e l e c t r i c point and treatment of t h i s toxin protein with concentrated ionized solutions (Block and Alexander 1960) could have added to s i g n i f i c a n t denaturation. This botulinus toxin has not been p u r i f i e d and the r e s u l t s obtained indicate no help f u l knowledge f o r further work. (35) (d) Clostridium botulinum Type D Sterne and Wentzel (1950) used media composed of unconcentrated corn-steep liquor (3.5% of t o t a l solids) with 0.5% glycerol and 0.1% sodium chloride, f o r the mass production of type D botulinus toxin. They developed a technique f o r the production of toxins i n carboys • with inserted cellophane sacs and obtained t o x i c i t i e s up to 130 x 10 6 mouse LD/mg N. P u r i f i c a t i o n was t r i e d with 0.-.-.40 saturated (NH^^ SO^ at pH 5.8 and a p r e c i p i t a t e was obtained which was redissolved (solution not specified) and reprecipitated with 25-30% saturated (NH^) 2 SO^ The preparation was e l e c t r o p h o r e t i c a l l y homogeneous and a molecular weight of 1,000,000 was reported. The p u r i f i e d 12 toxin gave a s p e c i f i c t o x i c i t y of 4 x 10 MLD/mg. N. Wentzel et a l (1950) reported that t h i s preparation turned out to be polydisperse when the d i f f u s i o n experiment was performed. The molecular weight indicated by these workers i s very high and the p o s s i b i l i t y e x i s t s that t h e i r molecular species was unhomogeneous and represents toxic molecules aggregated with non-toxic material. There are not enough data given to further analyse the procedure. The method i s based on " s a l t i n g out" with (NH 4) 2 SC>4 which i s quite a gentle treat-ment and decreases the p o s s i b i l i t y of denaturation (Neilands & Stumpf 1958). (36) (e) Clostridium botulinum Type E Gordon et a l (1957) obtained whole culture f i l t r a t e s by growing a type E culture i n 2.0% proteose peptone, 2.0% yeast extract (BBL) and 1.0% dextrin at 30° C f o r 7 days. The p r e c i p i t a t e was obtained from the whole culture by the addition of 95% ethanol to a f i n a l concen-t r a t i o n of 25% which also contained 1% Bentonite at -7° C. The p r e c i p i t a t e was brought up to 1/6 of the culture volume with water and s t i r r e d at room temperature for one hour. The preparation was further d i l u t e d to 1/4 culture volume with water, and 1 M CaClg was added to a f i n a l concentration of 0.075 M at pH 6.0. The p r e c i p i t a t e obtained af t e r s t i r r i n g f o r 2 hours at room temperature was discarded. The super-natant was treated with ethanol i n the previously described manner and the p r e c i p i t a t e was dissolved i n 0.08 M phosphate buffer at pH 6.0. F i n a l p r e c i p i t a t i o n was c a r r i e d out with ethanol and the p r e c i p i t a t e was dissolved in 0.02 M succinate buffer at pH 5.5. The preparation thus obtained was believed to be 410 times more pure than the o r i g i n a l whole culture with a t o x i c i t y of 19,000,000 LD5Q/mg<N. Sakaguchi and Sakaguchi (1959) worked on 48_hour, three times d i s t i l l e d water washed c e l l s which were grown on media composed of beef l i v e r infusion broth, brain heart infusion broth (Difco), V.F. broth, (peptic digest of beef and l i v e r ) and 0.5% glucose;;. The washed c e l l s were s t i r r e d at 37° C overnight in molar acetate buffer at pH 6.0. The extract was treated with (NHJ^SO. to a f i n a l concentration of 50-60% (37) saturation and dialyzed against water. The supernatant was l y o p h i l i z e d and p u r i f i c a t i o n was attempted with starch electrophoresis using acetate buffer at pH 6.0. The toxic f r a c t i o n i s o l a t e d by t h i s method contained a considerable amount of r i b o n c l e i c acid and had a t o x i c i t y of 6 x 10^ to 9 x 10 5 MLD/mgN. Gerwing et a l (1961, 1962) exploited the ion exchange chromatographic technique f o r the p u r i f i c a t i o n of ethanol prec i p i t a t e d toxins from cultures grown in cellophane sacs. The p r e c i p i t a t e was dissolved in 0.05 M sodium acetate at pH 6.0 in 1/20 of the o r i g i n a l culture volume. Ten m i l l i -l i t r e s of t h i s toxic preparation were then applied to columns packed with DEAE (Selectacel) c e l l u l o s e pretreated with 2 M .-sodium acetate solution at 4° C for 24 hours, washed with d i s t i l l e d water and then three times with 95% ethanol, dried, and suspended i n d i s t i l l e d water to make a homogeneous p a r t i c l e size s l u r r y . The toxin was eluted with ascending s a l t concentration s t a r t i n g from d i s t i l l e d water to 0.5M sodium acetate at a constant pH of 6.5. The toxin recovered was dialysed, l y o p h i l i z e d , resuspended in 0.05 M sodium acetate at pH 6.0 and rechromatographed i n the same way. The authors report that a ten-fold p u r i f i c a t i o n can be obtained by using t h i s method. The t o x i c i t y of the p u r i f i e d 5 preparation was 5.8 x 10 MLD/mg. N. The u l t r a c e n t r i f u g a l study of t h i s toxin showed two components with sedimentation constant §20 W = a n < * * n e toxic component being present i n the more rapidly moving boundary. (38.) Gordon and coworkers (1957) claimed that t h e i r toxic protein obtained aft e r repeated eMianol p r e c i p i t a t i o n s was 410 times more pure than the crude product which i n no way s a t i s f i e d any c r i t e r i a of purity. Sakaguchi and Sakaguchi (1959) also f a i l e d to obtain any s a t i s f a c t o r y r e s u l t s and the methods used for obtaining the toxins from cultures was more d i f f i c u l t , messy and less r e l i a b l e than the method used by Gerwing et a l (1961, 1962). Gerwing et a i (1961, 1962) f o r the f i r s t time used ion exchange chromatography f o r the p u r i f i c a t i o n of ethanol-precipitated toxin which was a s i g n i f i c a n t divorce from the methods used before. They obtained a p a r t i a l l y p u r i f i e d toxin component. Their treatment of chromatographic material before usage did not exploit the exchange potential of the r e s i n which led to poor-resolution i n the eluted f r a c t i o n s . In summary, various methods have been applied to pur i f y the A, B, C, D & E toxic proteins from the crude solutions from 1926 to date. Mainly, acid p r e c i p i t a t i o n , " s a l t i n g out" and more recently ion exchange chromatography were the basic techniques. The p u r i f i e d toxic proteins obtained by various workers had not s a t i s f i e d the c r i t e r i a of purity and were e s s e n t i a l l y unhomogeneous. (39) CHAPTER III  Preliminary Experiments The major work involved throughout t h i s research project centered on the solution to problems concerning the p u r i f i c a t i o n of the toxic protein produced by Clostridium  botulinum type E. The two immediate techniques to be developed were f o r the mass production of t h i s toxic protein with consistantly high y i e l d s and the concentration of toxin from the toxic f i l t r a t e s , maintaining the toxic material i n a soluble and b i o l o g i c a l l y active form. The f i n a l stages of the project were, concerned with the separation of a single protein species imparting the toxic a c t i v i t y . The work of Gerwing et a l (1961, 1962) was used as a guide at the s t a r t and the available information was exploited to f i n d the best and easiest method of solving these problems. The preliminary experiments performed imparted valuable information and formed the basis of the f i n a l successful technique. (a) Mass Production of Toxin Cultures were grown i n tubes of GPB1 broth f o r 24 hours at 37° C and were inoculated to a dialysate column (Fig. 1, Appendix 1) using GPB1 broth with 1.0% sodium t h i o g l y c o l l a t e and 2.0% dextrose (Appendix I I ) . A major problem of t h i s technique was the r i s k of contamination of the media during the incubation period which contributed to the production of low t o x i c i t y f i l t r a t e s . A modified apparatus was developed (Fig. 2, Appendix III) which was easy to handle, less prone to (40) contaminate the media and gave continuously high t i t r e toxic f i l t r a t e s . The dialysate cultures were harvested aft e r 80 to 90 hours growth at 30° C. It was r e a l i z e d l a t e r that i f growth was allowed to continue for 120 hours at 30° C in the dialysate columns using GPB1 media with 1% sodium t h i o g l y c o l l a t e and 0.5% dextrose, better and more consistant y i e l d s were obtained. This was previously shown by Dolman, C.E. (unpublished data). It was also observed that the a c t i v i t y of the dialysate toxins was decreased due to the cellophane sacs. (Sommer et a l (1926), also observed t h i s type of d e t o x i f i c a t i o n i n the case of Clostridium botulinum type A). It was noted that the cellophane sacs i f used a f t e r b o i l i n g i n O.lMVersene adjusted to pH 7.0 and washing several times with d i s t i l l e d water, had l i t t l e e f f e c t on the toxin production and dialysates of consistantly high toxic t i t r e s were obtained. (b) P u r i f i c a t i o n of the Concentrated Toxin The procedure of Gerwing et a l (1961, 1962) was t r i e d with ethanol pre c i p i t a t e d toxin which was suspended in 0.05 M sodium acetate solution at pH 6.5, for p u r i f i c a t i o n (Fig. 3). This procedure was t r i e d with various a l t e r a t i o n s such as the flow rate, column length and diameter r a t i o and variations in i o n i c strengths of eluting buffers. Although none of these experiments were completely successful i n obtaining the toxin in the pure state, important data was obtained i n some cases. These data w i l l be given to demonstrate the development of the f i n a l and successful technique. (41) c _, , , , , 200 300 +00 500 60O ML. EL UE/VT ^/O./S Chromatography of Botulinus Type E, ethanol prec i p i t a t e d toxin on a column packed with DEAE pretreated with 2 M sodium acetate solution. The graph shows the elutio n pattern of the preparation. (42) Procedure A: The column was packed with DEAE (Selectacel) C e l l u l o s e by the general method described by Gerwing et a l (1961) except that the DEAE c e l l u l o s e was ionized i n 2 M NaCl overnight , a f t e r which the column was washed with d i s t i l l e d water. Ten ml of the ethanol precipitated toxin was applied and e l u t i o n was c a r r i e d out with d i s t i l l e d water. A large amount of non-toxic material was eluted with the d i s t i l l e d water (Figure 4) although the toxin remained on the column. The inferences drawn from these r e s u l t s were: 1) there i s a water-soluble non-toxic component i n the toxic prepara-tions, and 2) these components are not adsorbed onto the column under the given conditions. Procedure B: In t h i s procedure, the DEAE (Selectacel) Cellulose was treated with 2 M NaCl solution overnight at 4° C before packing. The columns were packed under s l i g h t p o s i t i v e pres-sure. Ten ml of the ethanol pre c i p i t a t e d toxin was applied to the column and stepwise e l u t i o n was c a r r i e d out with 180 ml of d i s t i l l e d water and 1 M sodium acetate solution at pH 6.5 respectively (Figure 5). Further experiments were done to study the resolution and separation of various components, using eluting agents of constant molarity but d i f f e r e n t pH's (Fig. 6). The pH values of the e f f l u e n t s were also studied (Fig. 7). The l a s t experiment in t h i s series was done by using Dowex as the ion exchange material (Fig. 8). (43) NCM ro*ic — , i 1 ">o QOO too ML. ELUELNT FtG. -f Separation of a nontoxic and water soluble material from the toxic preparation of botulinus type E with chromatography on DEAE, 2 M NaCl pretreated columns. Elution was c a r r i e d out with d i s t i l l e d water. (44) 1-2--l.o- -i ^ 4. CV -2 - 9 -pH~VALU£S OPT/CAL VALUES' to si -6 - 6 ! I I I I ' I I I > i r*~' i NON TOXIC •0/STUCCO #to / 00 MAx.0-3>- — S ro 300 yn4*L/y,J) v . <*0<V TOKIt A T 6oo 290 300 /Vf/. £IVENT FIG. 6\ Stepwise elut i o n gradient of botulinus type E ethanol precipitated toxin on DEAE c e l l u l o s e (selectacel) columns, pretreated with 2 M NaCl at 4° C,' with d i s t i l l e d H O and 1 M Sodium Acetate solution at pH 6.5 /ml •, .1 ^ I I I I 8H foo i°*o "oo a oo i^oo f+oo rfoo ML ElUCHT Stepwise e l u t i o n gradient of botulinus type E, ethanol p r e c i p i t a t e d toxin, using 1 M sodium acetate solution at pH 6.5, pH 4.5 and pH 7.0 respectively, water-soluble non-toxic material i s previously removed with d i s t i l l e d HgO. (DEAE - c e l l u l o s e in 2 M NaCl i s used i n the column) (46) ML. FLUENT / r / 6 , 7 Stepwise elut i o n gradient of botulinus type E, ethanol pre c i p i t a t e d t o x i n ( l M Sodium Acetate solution at pH 6.5 and pH 4.5 i s used as eluent. The column i s packed with DEAE c e l l u l o s e , pretreated with 2 M NaCl at 4o C.) (47) MAX O.Tt—J-ML ELUENT P{Qt g Chromatograph of botulinus type E, ethanol preci p i t a t e d toxin on a column packed with Dowex pretreated with 2 M NaGl. El u t i o n i s c a r r i e d out with;., j d i s t i l l e d HQO and 1 M Sodium Acetate solutions (pH 6.5 and pH 4.5 respectively). (48) Procedure C: At t h i s stage, the washing of the packed columns with IN HC1 p r i o r to e q u i l i b r a t i o n , was introduced. It was f e l t that t h i s treatment was necessary to release a l l the extraneous material s t i c k i n g onto the column a f t e r the previous run. The column size chosen in t h i s case was 14 mm x 725 mm. DEAE (Selectacel) Cellulose was treated with 2 M NaCl over-night at 4° C and columns were packed under s l i g h t p o s i t i v e pressure. The columns were washed with IN HC1 and then with d i s -t i l l e d water t i l l the pH of the e f f l u e n t was at 5.5. Ten ml of the ethanol pre c i p i t a t e d toxin (suspended in 0.05 M sodium acetate solution at pH 6.5) was then applied to the column. Stepwise elut i o n was c a r r i e d out with the following solutions (applied i n the given order). 1. D i s t i l l e d water 225 ml. 2. 0.05 M sodium acetate solution at pH 6.5 675 ml. 3. 0.10 M " " M " " 200 ml. 4. 0.20 M " " " " " 285 ml. 5. 0.40 M " " " • - " 365 ml. 6. 0.60 M " " " " " 200 ml. The column was run at 4° C with an average flow rate of 10-15 ml/hr and 5.0 ml f r a c t i o n s were c o l l e c t e d . (Figures 9 and 10). Fractions 139-147 contained the toxic a c t i v i t y and were pooled. The b i o l o g i c a l a c t i v i t y of t h i s f r a c t i o n was then calculated as 6 5.0 x 10 MLD/ mgN. This toxin gave a sedimentation c o e f f i c i e n t of S W =1.7 and appeared to be a single component. The at) I O O a o o 300 jt**^ $oo goo . 700 .800 M L E L U fNT Ff&. 9. Chromatography of botulinus type E, ethanol p r e c i p i t a t e d toxin on DEAE c e l l u l o s e column, pretreated with 2M NaCl. The column i s washed with IN HC1 and d i s t i l l e d water a f t e r packing, and stepwise elution was c a r r i e d out with d i s t i l l e d H„0 and 0.05 M and 0.1 M sodium acetate solutions at pH 6.5 respectively. Fig.10. Schlieren photograph of u l t r a c e n t r i f u g a l behaviour of botulinus type E, ethanol pr e c i p i t a t e d and p u r i f i e d toxin. (51) material, however, appeared to be polydisperse because spreading of the boundary was more rapid than could be accounted f o r purely by ordinary d i f f u s i o n . (c) The preparation of Soluble Concentrated Toxin The toxic f i l t r a t e s were treated with 95% ethanol to a f i n a l concentration of 35% at -15° C overnight. The p r e c i p i t a t e obtained after centrifugation was suspended i n 0.05 M sodium acetate solution at pH 6.5. This suspension always contained a large amount of insoluble toxic material. This problem was investigated by suspending the toxic ethanol p r e c i p i t a t e in d i s t i l l e d water and then centrifuged at 0° C at 15,000 RPM for 30 minutes. Two f r a c t i o n s were obtained a f t e r centrifugation, i . e . the non-toxic supernatant and the toxic residue. The toxic residue was resuspended in d i s t i l l e d water to get r i d of a l l the non-toxic water-soluble components. Attempts were then made to obtain t h i s residue in a completely soluble form by suspending the residue i n various buffers and s a l t concentrations (Procedure A, Table 1). As i t appeared that at least 50% of the toxin was i r r e v e r s i b l y insoluble under the given conditions, further attempts to concentrate the toxin were c a r r i e d out using (NH ) SO as the i n i t i a l p r e c i p i t a t i n g 4 2 4 agent. (Procedure B, C, D, Table 2). Under these conditions, a high t i t r e soluble concentrated toxic preparation was obtained. The recovery rate was 75-80%. (52) Schematic procedures f o r the preparation of high t i t r e soluble concentrated toxin. Procedure A (unsuccessful) Toxic F i l t r a t e (dialysate toxin centrifuged and f i l t e r e d ) 95% ethanol t© a f i n a l concentration of 35% at - 15© C overnight. 1 Centrifuged at 0° C (15,000 RPM, 30") Pr e c i p i t a t e ^ Supernatant (Toxic) —^Suspended i n d i s t . H 0 (Non toxic 2 discard) I o Centrifuged at 0 C (15,000 RPM. 30") Residue 4^ Supernatant (Toxic) (Non toxic discard) Resuspended in d i s t i l l e d HO and c e n t r i -fuged at 0 C. Discarded the non toxic supernatant and resuspended i n 0.01 M sodium acetate buffer at pH 5.5. Centrifuged at 0° C. Residue -^Resuspended i n 0.5 M Sodium Supernatant (Toxic) Acetate Buffer at pH 5.5. (Low t o x i c i t y ) centrifuged at 0° C (15,000 RPM, 30") Residue Supernatant (Toxic) (Toxic) i Suspended i n 0.5 M NaCl solution. (53) Table 1 Preparation MLD/ml Toxic f i l t r a t e 3,000 Ethanol p r e c i p i t a t e suspended i n d i s t i l l e d water 30,000 ethanol supernatant 30 Water insoluble residue of ethanol p r e c i p i t a t e 20,000 Water soluble portion of ethanol p r e c i p i t a t e 30 Second water insoluble residue suspended i n 0.01 M sodium acetate buffer at pH 5.5. 10,000 Second water soluble portion of ethanol p r e c i p i t a t e 30 0.01 M sodium acetate buffer at pH 5.5. soluble portion 100 0.01 M sodium acetate buffer at pH 5.5. insoluble portion 3,000 to 10,000 suspended i n 0.5 M sodium acetate buffer at pH 5.5. 0.5 M sodium acetate buffer at pH 5.5. soluble portion 2,000 0. 5 M sodium acetate buffer at pH 5.5 in soluble portion suspended i n 0.5 M NaCl solution 2,000 (55) Table 2 Preparation MLD/ ml Toxic f i l t r a t e 1,000 33% saturated (NH )_SO ppt. suspended 4 2 4 in 0.01 M sodium acetate buffer at pH 5.5. 1,000 Supernatant 100 50% saturated (NH ) SO p r e c i p i t a t e sus-4 2 4 pended i n 0.01 M acetate buffer at pH 5.5. 3,000 Supernatant 100 60% saturated (NH ) SO p r e c i p i t a t e 4 2 4 suspended i n 0.01 M sodium acetate buffer at pH 5.5. 10,000 to 20,000 Supernatant 30 (54) Procedure B (unsuccessful) Toxic F i l t r a t e + Saturated (NH ) SO to a f i n a l concentration of 33% 4 2 4 saturation p r e c i p i t a t e d overnight at 4° C. Centrifuged at 4,500 RPM for .45 minutes at 0° C. / \ P r e c i p i t a t e Supernatant (Toxic) (Toxic) Procedure C (unsuccessful) Toxic F i l t r a t e + (NH ) SO to a f i n a l concentration of 50% saturation 4 2 4 -kept in fridge overnight. Centrifuged at 4,500 RPM for 45 minutes at 0° C. S \ P r e c i p i t a t e Supernatant (Toxic) (Toxic) Procedure D (successful) Toxic F i l t r a t e + (NH ) SO to a f i n a l concentration of 60% saturation 4 2 4 kept in fridge overnight. P r e c i p i t a t e Supernatant (Toxic) . (Non-Toxic) \ Suspended i n 0.01 M Sodium Acetate Buffer at pH 5.5. Centrifuged at 4,500 RPM f o r 45 minutes / a t 0 0 c- V Residue Supernatant (Non-Toxic) (Toxic) (56) F i n a l l y , a f t e r obtaining the high t i t r e concentrated soluble toxin, attempts were made to pur i f y t h i s material by using the following method. DEAE (Selectacel) Cellulose pretreated with 2 M NaCl solution overnight at 4° C was packed into columns (column si z e 14 mm. x 725 mm) and was washed with 1 N HC1. Ten ml of the (NH ) SO pr e c i p i t a t e d toxin (dissolved i n 4 2 4 0.01 M sodium acetate buffer at pH 5.5.) was applied to the column, equilib r a t e d with the same buffer. E l u t i o n gradient was then t r i e d with 0.01 M sodium acetate buffer at pH 5.5. and 1M NaCl. Sodium chloride provided a steady increase in molarity (from 0. to 0.5 M) in a constant background of buffer at pH 5.5. The elut i o n was c a r r i e d out at room temperature as well as the packing of the column, washing and e q u i l i b r a t i o n (Fig. 11a, l i b ) . The encouraging r e s u l t s observed here were further investigated and formed a basis for the successful experimental work. An analogous run with columns equilibrated i n pH 4.5 buffer rather than pH 5.5. was ca r r i e d out. The r e s u l t s (Fig. 12) showed much better resolution of toxic material-and subsequent runs were made using buffer at pH 4.5 for both e q u i l i b r a t i o n and e l u t i o n . (57) • i too 200 ht. ElUE/VT F i C l l b . •now to*<c 1M - N a O « . —OOSM SODIUM ffCET&TE pH «•-y . — E t V T ' O K C « f i D I £ W T — * — T — 100 1 loo ML. EtUENT -%— 3oo F»"G.||*. Chromatography of botulinus, type E, (NH4)2,S04 preci p i t a t e d toxin on a DEAE c e l l u l o s e column. (58) .eg-1 ,to*<C •06-r -a. Of o CO o oo oii-.o%-W-N/aCft—<H«1 SoDtUM ACETATE p H l ^ f ELUTfOJV GRfVblCOfT- > too I ML ELUEIVT 30o Fi& a Chromatography of botulinus, type E, (NH ) oS0. preci p i t a t e d toxin on a DEAE c e l l u l o s e column. (59) CHAPTER IV  Materials and Methods The s t r a i n of Clostridium botulinum type E used in t h i s work was i s o l a t e d by Nakamura et a l (1956) from an outbreak of fish-borne botulism in Hokkaido, Japan. This s t r a i n was toxigenic, non-proteolytic, gas producing and named as the Iwanai s t r a i n of Clostridium botulinum, type E. This s t r a i n gave a consistant t o x i c i t y of 1,000 - 3,000 MLD/ml when grown in culture tubes of GPB1 medium. (Appendix I I ) . The s t r a i n , maintained as a pure culture in GPB1 medium, was obtained through the courtesy of Connaught Laboratories, Western D i v i s i o n , University of B r i t i s h Columbia, and the stock cultures were p e r i o d i c a l l y checked on Brain Heart Infusion agar plates (Appendix IV). Tubes containing 15 ml of GPB1 containing 1% t h i o -g l y c o l l a t e and 2% dextrose added a s e p t i c a l l y as 50% dextrose were used for the maintainance of stock cultures as well as f o r the growth of the seed cultures. The cultures were grown at 37° C for 24 hours. GPB1 media with 1% t h i o g l y c o l l a t e (DIFCO) was used in large volume toxin production. F i f t y percent dextrose was added to the GPB1 media a f t e r autoclaving to a f i n a l concentration of 0.5%. (60) (a) Toxin Production Large volume toxin production involved the use of a d i a l y s i s apparatus (Vinet and Fredette 1951) modified in t h i s laboratory in order to cut down b a c t e r i a l contamination of media from the apparatus as well as from outside (Appendix I, F i g . 1). The cultures were grown in the cellophane sacs (Appendix III, Fig.2) for f i v e days at 30° C with d a i l y changes of media. The seed culture of 30 ml in two GPB1 tubes was incubated at 37° C for 18-24 hours p r i o r to ibnoculation. (b) Toxin P r e c i p i t a t i o n The Seitz f i l t e r e d dialysate toxins were treated with saturated (NH^^SO^ to a f i n a l concentration of 60% saturation for 10-16 hours at 4° C. The toxic p r e c i p i t a t e , obtained a f t e r centrifuga-tion of the f i l t r a t e at 4,500 RPM f o r 45 minutes at 0° C. was suspended i n 0.01M sodium acetate buffer at pH 5.5., and the volume brought to approximately 1/20 the o r i g i n a l f i l t r a t e volume. This material was dialysed against 0.01 M sodium acetate buffer at pH 5.5. overnight. The insoluble non-toxic material was removed by centrifugation. (c) Toxin P u r i f i c a t i o n DEAE (Selectacel) c e l l u l o s e synthesized and described by Peterson et a l (1956) was used as the ion exchange material. This material was suspended i n 2.0 M NaCl solution (10 gms of the cell u l o s e per l i t r e of 2 M NaCl solution) and kept at 4° C. Columns of varying lengths with diameter to length r a t i o of 1/20 were packed under gravity flow at room temperature (Appendix V). The columns were then washed with 1 N HC1 and (61) equilib r a t e d with O.OlMi. Sodium acetate buffer at pH 4.5 at room temperature. Five ml of the concentrated toxin were then applied to the column and eluted f r o n t a l l y with 0.01 M sodium acetate buffer at pH 4.5 at room temperature. Fractions of 5 ml were c o l l e c t e d on an automatic f r a c t i o n c o l l e c t o r model V-10 (Gilson Medical E l e c t r o n i c s ) . (d) Treatment of D i a l y s i s Paper The d i a l y s i s paper used i n the production of toxins as well as in d i a l y s i s was pretreated with b o i l i n g 0.1 M Versene adjusted to pH 7.0 for f i v e minutes and washed several times in d i s t i l l e d water. (e) Assay Technique S e r i a l decimal d i l u t i o n s of the toxin were made i n 0.85% of saline solution depending on the expected t o x i c i t y andihnoculated i n t r a p e r i t o n e a l l y into mice of weight between 15-25 gms with the use of #26 needles. Rough t i t r a t i o n s were c a r r i e d out by using one mouse per dose while f i v e mice per dose were used f o r an accurate assay. The smallest dose which k i l l e d the animal in 48 hours was calculated as the MLD/ml. (f) A n a l y t i c a l Techniques Various f r a c t i o n s c o l l e c t e d during the f r o n t a l elution were analysed f o r 280 mja and 260 mja absorbing material on a Beckman Model DU spectrophotometer using a B.D.C.C. quartz curvette. Sodium acetate buffer of 0.01 molarity and pH 4.5 was used in the standard c e l l . (62) Total nitrogen was determined by using the semi-micro Kjeldahl technique (Rabat & Mayer 1948). B o i l i n g chips used i n t h i s technique were coated with'selenium which replaced copper-sulphate as the c a t a l y s t f o r the digestion process. A Beckman/Spinco Model E a n a l y t i c a l u l t r a c e n t r i f u g e and a SpincoModel H electrophoresis d i f f u s i o n apparatus were used for analysis of the material prepared in t h i s work. The amino-acid analysis of the p u r i f i e d component was c a r r i e d out on an automatic amino-acid analyser b u i l t by Reichmann (Federal A g r i c u l t u r a l Research Station, Vancouver, B r i t i s h Columbia) based on the Moore & Stein (1954, 1956) technique. (g) Preparation of Buffer solution Various buffer solutions used in t h i s work were prepared by c a l c u l a t i n g components with Henderson-Hasselbalch equation (Appendix VI). (63) CHAPTER V  Results (a) Degree of p u r i f i c a t i o n . The a c t i v i t y per mg.N of the various toxic preparations i s summarized i n table 3. It can be seen that a two thousand-fold increase in s p e c i f i c a c t i v i t y was obtained during p u r i f i c a t i o n . Table 3. No. Toxin Preparation MLD/mgN Yi e l d % 1. Ori g i n a l Toxic F i l t r a t e 3.2 x 10|) 100 2. Ammonium sulf a t e precipi-tate, a,fter d i a l y s i s and centrifugation 4.9 x 10 4 80 3. Pure preparation 7.5 x 10 6 50 (b) Elution Chromatography. Ammonium sulfate precipitated toxin, dissolved i n 0.01M sodium acetate buffer at pH5.5. and eluted with same buffer at pH4.5, came out f r o n t a l l y . After the passage of 100 ml. of the eluent, two peaks were obtained; the former being extremely toxic and the l a t e r peak non-toxic (Fig. 13). The major portion of the non-toxic material remained adsorbed onto the column and would be removed i n one step with IN HC1 (Fig. 14) or more slowly using a gradient or stepwise e l u t i o n . Chromatographed, reprecipitated toxin gave a single peak under the same set of conditions indicating a high l e v e l of purity.(Fig.15) F i n a l pure (64) oas Mo so Ho M L ELUE.WT piG. 13. Chromatography of botulinus type E, (NH^) 2SO4 prec i p i t a t e d toxin on a DEAE c e l l u l o s e column, pretreated with 2M NaCl, washed with IN HC1; equ i l i b r a t e d and eluted with 0.01 M sodium acetate buffer at pH 4.5. (65) fJOr-i TOUC PEAK ML.TLUZNT FIG. )H Chromatography of botulinus type E (NH^gSO pr e c i p i t a t e d toxin (same conditions as i n F i g . 13). This figu r e shows the el u t i o n of the adsorbed non-toxic material with IN HC1 from the column a f t e r e l u t i o n of the toxin. (66) M L . E.LUEIVT FlG, lix Rechromatography of botulinus type E, chromatographed and reprecipitated toxin. The figure shows a single sharp peak. (67) 6 preparation contained 7.5 x 10 MLD/mgN and the y i e l d was 50%. (c) U l t r a - V i o l e t adsorption spectrum. The p u r i f i e d toxin showed a UV absorption spectrum t y p i c a l of most proteins < with maximum absorption at 277 m/4L (Fig. 16). The absence of any subsidiary absorption at other wave lengths has indicated at least the lack of non-protein impurities. (d) U l t r a c e n t r i f u g a l Analysis. The u l t r a c e n t r i f u g a l analysis showed the presence of a single, apparently homogeneous component in an a r t i f i c a l boundary c e l l , run at 59780 RPM. Schlieren photographs were taken at sixteen minute i n t e r v a l s . (Fig.17). The sediment c o e f f i c i e n t was calculated as S20^ = I - 7 0 - The shadow of a rapidly moving component was also observed which could indicate a low percentage of impurities or the presence of aggregated molecules. (e) Electrophoretic analysis. The behaviour of the material was studied extensively at pH4.5 in a Spinco - Model H electropho-r e s i s d i f f u s i o n apparatus and showed the presence of a large —5 single homogeneous component with a mobility of +5.66 x 10 / 2 cm /Vol. sec. (Fig. 18). The r e s u l t s also showed a small, immobile peak, which disappeared rapidly and was assumed to be the ^ boundary. (g) D i f f u s i o n properties. A Spinco Model H Electrophoresis D i f f u s i o n apparatus was used for d i f f u s i o n properties and the 2 d i f f u s i o n c o e f f i c i e n t was calculated at 4.8 x 10""7 cm /sec. at 1° C and 8.87 x 10~ 7 cm 2/sec. at 30° C over a period of 72 hours. (68) Pig. 17. Ultracentrifugal analysis of botulinus type E, purified toxin. Schlieren photographs were taken at sixteen minute intervals. Ultracentrifuge was run at 59780 RPM. (70) Fig. 18. Electrophoretic behaviour of botulinus type E purified toxin. (71) hours. (g) Molecular weight determination. The molecular weight was calculated to be 18,600 with the following equation: M = 20 D 2 0(1-VP) M = Molecular weight. R = Gas Constant T = Absolute temperature. S 20 W " Sedimentation c o e f f i c i e n t at 20° C. V = P a r t i a l s p e c i f i c volume. P - Solution Density. and D = D i f f u s i o n c o e f f i c i e n t at 20° C. 20 (h) Amino Acid Analysis. The p u r i f i e d toxin was hydrolysed in vacuo in 6N Hcl at 106° C f o r 18 hours. HC1 was removed by freeze—drying and the amino-acid preparation was washed three times i n d i s t i l l e d water and flash-evaported at 42° C. These preparations were run on Moore and Stein (1954) columns in order to obtain the amino acid r a t i o s . Tryptophane, always destroyed under these conditions, has not as yet been calculated. Methionine and cysteine, which must be calculated as methionine, sulfone and c y s t e i c acid i n order 1t;o give accurate r e s u l t s on the Moore 8s Stein (1954) columns,have not yet been evaluated although performic acid oxidations have been c a r r i e d out on hydrolysates. Results for the remaining amino acids are shown in Table 4. (72) TABLE 4 The amino acid analysis of type E toxin. Amino Acid Micromoles Molecular per ml. mg/ml r a t i o Lysine 0.137 0.0176 9 Hi s t i d i n e 0.0152 0.0021 1 Arginine 0.041 0.0064 3 Aspartic acid 0.246 0.0283 16 Threonine 0.072 0.0073 5 Serine 0.112 0.0098 8 Glutamic acid 0.166 0.0214 11 Proline 0.095 0.0092 6 Glycine 0.180 0.0106 12 Alanine 0.093 0.0066 6 Valine 0.089 0.0088 6 Isoleucine 0.147 0.0166 10 Leucine 0.144 0.0163 10 Tyrosine 0.0517 0.0084 4 Phenylalanine 0.074 0.0109 5 (73) CHAPTER VI  Discussion The basic aims of t h i s research project, i . e . , the p u r i f i c a t i o n and characterization of botulinus type S toxin, have been successfully accomplished. The problems encountered i n the process were mainly the ones connected with the production, concentration and p u r i f i c a t i o n of t h i s toxin. The method of producing toxins in d i a l y s i s sacs introduced by Vinet and Fredette (1951) and modified i n t h i s laboratory (Appendix III, F i g . 2) was used, and consistent y i e l d s of 3,000 to 5,000 MLD/ml of toxic f i l t r a t e s were obtained. S i m p l i f i c a t i o n of the basic technique has s i g n i f i c a n t l y cut down on media contamination which was one of the major hurdles i n obtaining good y i e l d s of toxin. The i n a c t i v a t i o n of botulinus toxins during d i a l y s i s was noted by Sommer et a l (1926, 1928) working with Type A. No explanation was given at that time for t h i s loss of a c t i v i t y . Decrease in t o x i c i t y of botulinus type E toxin was also noted during d i a l y s i s i n t h i s laboratory, and the assumption was made that the toxin was i r r e v e r s i b l y destroyed by the surface active compounds present on the cellophane sacs. Pretreatment of the cellophane sacs with 0.1 M Versene at pH7.0 a l l e v i a t e d the negative e f f e c t of the surface active compounds with the r e s u l t that l i t t l e t o x i c i t y was l o s t during d i a l y s i s . It was confirmed that the Versene pretreated cellophane sacs used i n the production of dialysate toxins appreciably s t a b i l i z e d the toxic y i e l d s . (74) The dialysate toxins are superior to those produced in f l a s k s as the larger media components are not present. In p u r i f i c a t i o n studies, such as those undertaken here, i t i s imperative to s t a r t with f i l t r a t e s containing as l i t t l e as possible extraneous matter, p a r t i c u l a r l y proteins. Indeed, the behaviour of f l a s k toxins was markedly d i f f e r e n t , in ion-exchange chromatography, from dialysate toxins, under the same set of conditions. (Fig. 19). Gordon et a l (1957) worked on the f l a s k toxins which contributed to the variance of the i r r e s u l t s from the ones obtained here. The method used by Sakaguchi and Sakaguchi (1959, 1963, 1964) of extracting type E toxins from washed c e l l s has possible disadvantages to the methods used here. These workers obviously have had considerable d i f f i c u l t y in obtaining t h e i r toxic components i n a state free from nucleic acids and nuclear-proteins. It i s possible that the toxin, before i t i s released by autolysis from the c e l l s , i s linked or aggre-gated i n some way to such c e l l components. The superiority of (NH4)gSO over ethanol as a pre-c i p i t a t i n g agent was noted. Ethanol pr e c i p i t a t e s were essen-t i a l l y insoluble in the various e l e c t r o l y t e s t r i e d , while (NH ) SO pr e c i p i t a t e s were e a s i l y soluble. Ethanol may bring about p a r t i a l denaturation during p r e c i p i t a t i o n r e s u l t i n g i n aggregation. This type of denaturation may be attributed to the lack of adequate e l e c t r o l y t e s and to pH's beyond the i s o -e l e c t r i c point (Alexander and Block, 1960). Ammonium sul f a t e , on the other hand, caused no appreciable denaturation and (75) LEfrEIYD. F L f t S K T O X W S B l R L N S f t T E TOXIN t i 25o Jo is ML. tLUE/VT F|G. 19. Comparative chromatographic behaviour of dialysate and f l a s k toxins of botulinus type E, p r e c i p i t a t e d and eluted under s i m i l a r conditions. (76) aggregation. Sodium sulfate may also be used i n p r e c i p i t a t i o n ; the advantage being that i t does not impart any ammonia to the preparation. The chromatographed toxic preparation contained 1,000 times more t o x i c i t y per irig.N than the crude toxic p r e c i p i t a t e s . The toxic protein was eluted f r o n t a l l y while the impurities remained adsorbed onto the column. (Fig. 14). Sodium acetate buffer solutions of 0.01 molarity and of pH's ranging from 3.8 to 4.8 successfully eluted the toxic protein. A number of other pH's were t r i e d as well as elution gradients which did not y i e l d the same r e s u l t s . (Figs. 11a, l i b , 12, 20). Diameter to length r a t i o of the column was found to be very important. It was also observed that the molarity of the buffer solutions used did influence the r e s u l t s . The pH at which prec i p i t a t e d toxins were resuspended was c r i t i c a l with respect to successful e l u t i o n of pure toxin. Toxic preparations in buffers of pH's other than 5.5 when added to column, either did not give f r o n t a l e l u t i o n or gave f r o n t a l peaks containing impurities. DEAE (Selectacel) c e l l u l o s e was used as an ion exchange material f o r chromatography of the concentrated toxin. This c e l l u l o s e i s an anion exchanger and 20-40% of the CHgOH and/or OH groups on the c e l l u l o s e molecule form an ether l i n k -age when treated with dichloro-diethyl amino ethyl hydrochloride with the removal of the ions from the ethyl group (Appendix VIII). (78) This c e l l u l o s e was suspended i n 2M NaCl and kept at 4° C. (Fig.21). Columns a f t e r packing were treated with IN HC1 t i l l the pH was less than 0.01. This treatment removed excess Na and replaceable OH groups, imparting to the c e l l u l o s e excess H +. Sodium acetate buffer during e q u i l i b r a t i o n takes up H+ and Cl"ions and the c e l l u l o s e must be converted into the / c e l l u l o s e CI~, CH COO form at pH 4.5, as the t o t a l removal of H+ and Cl ions would r e s u l t in a basic pH. This combination of ?1 and CH COO ions on the c e l l u l o s e builds up appropriate conditions 3 in sieving, adsorption and ion exchange, which allows the toxin to be f r o n t a l l y eluted while non-toxic material remains adsorbed on the column. This non-toxic material can be removed from the ? columns by treatment with IN HC1 (Fig. 14). Excess H* decreases the adsorptive capacity of DEAE c e l l u l o s e s ; t h i s i s why adsorp-ti o n under pH 4.0 i s due to Vander Waal's forces and hydrogen bonding. (Boardman and Partridge, 1955). This explains to a c e r t a i n extent the i n a b i l i t y of DEAE c e l l u l o s e to give r e s u l t s below pH 3.8. It can be safely emphasized here that the number of cl"* and CH^COO ions present on t h i s ion exchange material i s important i n the e l u t i o n of type E botulinus toxin i n the pure form. E l u t i o n patterns f o r various Clostridium botulinum type E s t r a i n toxins have shown s i m i l a r behavior on DEAE (Selectacel) c e l l u l o s e treated i n t h i s way. A possible improvement torroutine ion exchange chromatography for the p u r i f i c a t i o n of proteins i s currently being developed i n t h i s laboratory. It i s hoped that the p r i n c i p l e s of ion exchange and those of electrophoresis may be combined and exploited maximally (Appendix VII, Fig.22). (80) The c r i t e r i a of purity used s a t i s f y the contemporary requirements f o r the i n t e g r i t y of the p u r i f i e d preparation. C r y s t a l l i z a t i o n was not applied, as t h i s criteriion i s unr e l i a b l e . C r y s t a l l i n e proteins do not necessarily contain one protein species as two protein species may be isomorphous and are constituents of one type of c r y s t a l s . A d i f f e r e n t set of c r y s t a l s may also be formed under another set of conditions. Emphasis was l a i d on: 1) b i o l o g i c a l a c t i v i t y per unit N, 2) paper electrophoresis. 3) column-chromatography, 4) u l t r a c e n t r i f u g a l analysis and 5) d i f f u s i o n electrophoresis, as the c r i t e r i a f o r purity. The p u r i f i e d toxic protein gave a t y p i c a l Gaussian curve i n the schlieren o p t i c a l systems used, indicating the presence of a single major component i n the electrophoretic and u l t r a c e n t r i f u g a l studies. An extremely small percentage of impurity was apparent which took the form of a rapid l y sedimenting small shoulder which appeared sixteen minutes after f u l l speed was attained in the ultra c e n t r i f u g e . This was supposed to consist of aggregated toxic molecules and to be insignificantdue to i t s low concentration i n comparison to the t o t a l protein present. A small immobile peak having the behavior of £ boundary, was seen i n the electrophoretic analysis which did not (81) move when the sample was run at pH 4.5 and pH 5.5., and d i s -appeared a f t e r about t h i r t y minutes. If t h i s immobile boundary peak had been due to a contaminant rather than to an interface reaction, one would have expected to have observed at least a degree of mobility at one of the pH's used for the investiga-t i o n . Paper electrophoresis and ion exchange column chromato-graphy indicated the presence of one single component i n the toxic preparation. Sakaguchi et a l (196'4) recently hypothesized the molecular weight of type E toxin to be larger than 200,000. They did not support the r e s u l t s reported by Gerwing et a l (1962). These authors joined the galaxy of previous workers who reported very high molecular weights for other botulinus toxins (Kegeles, 1946; Putnam et a1,1946, 1948; Lamanna and Glassman, 1947; Wagman, 1954). The molecular weight determina-ti o n of ethanol as well as ammonium sulfate precipitated toxic preparations was shown to be less than 20,000 (sedimentation c o e f f i c i e n t for both the toxic preparations i n the ultracentrifuge being 1.7) in t h i s laboratory. The data obtained i n t h i s work, combined with the c r i t i c a l analysis made of previous research done on the p u r i f i c a t i o n of botulinus toxins, indicate that the high molecular weight concept widely accepted for these substances, i s probably erroneous. (82) S U M M A R Y 1. A method fo r the p u r i f i c a t i o n of the toxic protein produced by botulinus type E, s t r a i n Iwanai, has been developed and a preparation has been obtained which appears to be homogeneous under u l t r a c e n t r i f u g a l and electrophoretic analysis. 2. The sedimentation c o e f f i c i e n t of the pure prepara-ti o n i s S°2QW = 1.7 and the molecular weight i s calculated to be 18,600. 3. Amino acid analysis of the preparation has been c a r r i e d out, although values f o r tryptophane, methionine and cysteine are yet to be calculated. 4. Molecular structure of the DEAE (Selectacel) c e l l u l o s e has been explained and an apparatus i s included i n the appendices which w i l l be used f o r future p u r i f i c a t i o n work. 5. A modified dialysate apparatus has been b u i l t f o r the mass production of botulinus toxins. (83) APPENDIX I D i a l y s i s apparatus for the mass production of Clostridium  botulinum toxins was f i r s t reported by Vinet and Fredette i n 1951. The apparatus consisted of a pyrex tubing (6.4 x 120 cm.) with two ends made f o r #9 rubber stoppers. This tube had two outlets, one on ei t h e r side approximately six inches from each end. The outlet on the lower end was connected to two (six l i t r e ) pyrex glass bottles through a T-tube. A loose cotton plug was placed i n the upper outlet which served as a pressure release from the glass tubing. Cellophane tubing was inserted into the glass and fixed at either end with rubber stoppers. The saline and innoculum was introduced through T-tube connection i n the lower stopper, the other outlet of which was used f o r sampling and harvesting. A piece of cotton wool plugged glass tubing was inserted through the upper stopper which allowed the escape of gas produced during b a c t e r i a l growth. The glass tube with cellophane sac, saline, and d i s -card bottles with respective rubber tubing connections was s t e r i l i z e d f or 1-1/2 hours under 15 pounds pressure. The media bott l e was s t e r i l i z e d separately f o r 1 hour at 15 pounds pres-o sure and 230 F. The apparatus was then set up in an upright position in the 30° C incubator. Eight hundred ml of the s t e r i l e s a line was added to the cellophane sac and the media was pumped with negative pressure into the outside of the (84) cellophane sac. A l l the outlets were clamped. The medium was allowed to d i f f u s e through the cellophane sac for overnight and then 40 ml. of a 24 hours' seed culture was ihnoculated through the saline b o t t l e . Complete changes of mediumwere made every 24 hours and the cultures were harvested after 80 to 90 hours' incubation at 30° C. MEDIUM 80TTLE. (85) DISCARD eoTTt-e _LL SftUNE J BOTTLE CELLOPHANE SAC •3UU.YSIVTE. c o L l f l W -SftMfLt TUBE Fl&.l. Dialysate apparatus for the mass production of botulinus, type E toxin. (86) APPENDIX II  Glucose Peptone Beef Media This medium was used f o r tube cultures with added meat p a r t i c l e s and f o r the mass production of dialysate and f l a s k toxins. F i n e l y ground beef (round steak) was infused over-o o night in tap water at 4 C, and then at 65 C for 45 minutes in the top of a double b o i l e r . Meat p a r t i c l e s were taken off by s t r a i n i n g through "cheese c l o t h " muslin and the f i l t r a t e was allowed to b o i l for 3 minutes. The infusion was then cooled to approximately 60° C and f i l t e r e d through a Whatman #1 f i l t e r paper i n order to obtain f a t and meat f i b r e free preparation. The volume was made up to the o r i g i n a l with tap water and the peptone and s a l t constituents per l i t r e were added as follows: NaCl 5 gms. Proteose peptone (DifCO) 10 gms. Na HP 0 .12 H 0 2 gms. 2 4 2 Sodium t h i o g l y c o l l a t e 1 gm. The pH was adjusted to 7.8 a f t e r d i s s o l v i n g the ingredients. F i f t e e n ml. of the medium and 1 gm. of a i r dried meat p a r t i c l e s were dispensed into 20 x 170 m.ra. pyrex tubes, plugged with non-absorbent cotton, and s t e r i l i z e d i n the autoclave f o r 30 minutes at 15 pounds pressure and 230 F. The medium f o r the production of dialysate toxins was dispensed i n s i x l i t r e q uantities into 10 l i t r e aspirator, plugged and autoclaved f o r 90 minutes at 15 pounds pressure and 230°F. F i f t y percent ( 8 7 ) dextrose solution was s t e r i l i z e d at 230° F and 15 pounds pressure, and added to tube medium (2%) and bo t t l e medium (.05%). (88) APPENDIX III D i a l y s i s apparatus f o r the mass production of botulinus toxins'' Table 5 S. No. Name Parts of Dialysate Apparatus  Description Glass tubings (a) 5.6 x 90 cms; thick pyrex glass annealed, dialysate column. (b) 6.5 x 75 mms. Quantity 1 7 2. Brass plates (a) outer brass plates, 7.0 cms diameter with 4.45 cms. diameter empty space, 6.5 mms. thick, and two holes on eithe r side (6.5 mm. diameter). 2 (b) inner brass plates, rectangu-l a r (7.0 x 2.5 ems) with three holes (one hole i n the centre and two on eithe r side of 6.5 mm. diameter). 2 3. Rubber corks 4. Pressure Rubber Tubings (a) outer rubber corks #7 (both have 4.45 cms diameter, hole i n the centre. The cork used on upper side of the columns have two extra (6.5 mm. diameter) holes on either side of the ce n t r a l one. The cork used at the lower end have one hole (6.5 mm. diameter) on one side of the ce n t r a l one. 2 (b) inner rubber corks #4 with holes i n the centre (6.5 mm. diameter) 2 (c) rubber cork f o r the discard b o t t l e #5 with two holes (6.5 mm. diameter) i n the centre and 25 mms. apart. (d) rubber cork #2 with 6.5 mm. diameter hole i n the centre. 1 (a) Six inches long f o r connect-ing media and toxin outlets 2 * Modified from the o r i g i n a l of Vinet and Fredette (1951) Name 5. Aspirators (89) Table 5 (continued) (b) Two feet long f o r connecting media and saline i n l e t s 2 (a) Media b o t t l e , 10 l i t r e s capacity 1 (b) Media discard b o t t l e , 8 l i t r e s capacity 1 (c) Saline b o t t l e , two l i t r e s capacity 1 6. Cellophane Sac 7. Brass rods 8. Screws 9. Stopcocks 3-1/2 feet long, versene pretreated 1 3-3/4 feet long, 6 mm. diameter (a) For screwing the inner plates (b) For screwing the outer plates 4 4 4 After the assembly, the dialysate apparatus (Fig.2) i s autoclaved according to the s p e c i f i c a t i o n s given i n Table 6, Name Assembled Dialysate Columns Medium Bottle Saline Bottle Discard Bottle 50% Dextrose Solution Table 6 Temperature 230° F 230° F 230° F 230° F 230° F Pressure in Time i n Minutes pounds/cm 2 120 90 60 30 15 15 15 15 15 15 Various connections are made a s e p t i c a l l y a f t e r auto-cl a v i n g . Eight hundred ml of saline i s then passed into the cellophane sac and the media i s f i l l e d outside up to the inside (90) l e v e l of s a l i n e . After 18-24 hours, media i s discarded and the dialysate i s innoculated with 24 hours tube cultures; two tubes of which are added a s e p t i c a l l y to the sa l i n e bottle and passed into the cellophane sac. The outside of the sac i s again f i l l e d with medium which i s changed a f t e r every 24 hours fo r the f i r s t three days while only a half change i s made during the l a s t two days of incubation. The toxin i s harvested a f t e r incubation f o r f i v e days at 30° C. (91) KPPEWD1V I E T o S f l u ^ E . BOT7JLE. OQ QC IS o > STOP COCK ^To MESlft BOHtE. j / M f f OUTLET * ME3>1B INLET OUTER S R f l S S PtATE •* OUTER CORK * SRUNE. (WLtT SUPPORTING, flgASS fffl> ^ miopnm SAC ^ . p y / f f X GLASS :D»W.T*AT£ c c M M / v • O U T L E T > T0XJI/V OUTLET » TO tf£J>/A DISCARD BOTTLE Modified dialysate apparatus f o r the mass production of botulinus type E toxin. (92) APPENDIX IV  Brain Heart Infusion Agar The dehydrated medium was rehydrated by d i s s o l v i n g 37 gms in 1,000. ml of d i s t i l l e d water. This medium with 1.5% agar was autoclaved for 15 minutes at 15.pounds pressure and 230° F. Eighteen ml. of the cold (45°C) medium was a s e p t i c a l l y poured into each plate. Formula In g r e d i e n t s / l i t r e Calf brains, infusion from 200 grams Beef heart, infusion from 250 grams Proteose Peptone, Difco 10 grams Bacto-dextrose 2 grams Sodium Chloride:; 5 grams Disodium Phosphate 2.5 grams (93) APPENDIX V Preparation dfthe ion exchange chromatographic column used for the el u t i o n of Clostridium botulinum type E, s t r a i n Iwanai.toxin. DEAE Cellulose (Selectacel) i s obtained commercially in the OH form. Ten grams of t h i s material was suspended i n one l i t r e of IN HCl for about one hour at room temperature af t e r which the HCl was removed by negative pressure. The material was washed three times in d i s t i l l e d water and a homogeneous suspension was then made i n IN NaOH. This process was repeated three times and the material was f i n a l l y sus-pended i n one l i t r e of 2M NaCl and kept at 4° C. The acid cleaned and d i s t i l l e d E^Q washed glass columns with sintered glass at t h e i r lower ends were p a r t i a l l y f i l l e d with 2M NaCl solution to which the c e l l u l o s e prepara-ti o n was added, and the columns were packed by gravity flow the subsequent addition of c e l l u l o s e and 2M NaCl. The packed columns were then washed with IN HCl t i l l the pH dropped to 0.01. The columns were then ready for e q u i l i b r a t i n g arid f o r further use, etc. (94) APPENDIX VI Henderson-Hasselbalch Equation pH = pka + (log s a l t ) acid where pH = hydrogen ion concentration, i o n i z a t i o n constant, s a l t concentration of the buffer acid concentration of the buffer pKa = s a l t = acid = pKa values equals pH when the acid i n solution i s half neutralized. The pH i s about one unit lower when 10% acid i s neutralized and pH i s one unit higher when 90% acid i s neutralized. One can pick buffers which w i l l maintain the pH r e l a t i v e l y constant in the desired range and the choice i s based on t h e i r pKa values. (95) APPENDIX VII ELECTRO-CHROMATOGRAPHIC APPARATUS This apparatus i s being devised i n t h i s laboratory.for the separation of proteins and polypeptides. The p r i n c i p a l of electrophoresis and ion exchange chromatography i s combined i n order to achieve better and fa s t e r analysis (Fig. 22). The apparatus consists of a chromatographic column (ion-exchange material packed i n s i d e ) . This column has two buffer i n l e t s and two buffer outlets on either side with a specimen i n l e t i n the centre. Two electrodes are f i t t e d on either side and the whole apparatus i s inserted i n an outer thick pyrex glass tube. The specimen i s applied i n the centre and the apparatus i s placed h o r i z o n t a l l y on two f r a c t i o n c o l l e c t o r s . After the specimen i s absorbed; an adequate current p o t e n t i a l i s passed through the column. The eluti o n i s then c a r r i e d out with an adequate buffer by running the column on one fractio n a t o r and cl o s i n g a l l the other outlets and i n l e t s . Then the column i s run on the other fractionator; the previous outlets being closed now. The d e t a i l s of t h i s apparatus are yet to be worked out as i t i s s t i l l i n the preliminary stages of development. (96) TO f£f\dTKMQTOR%€ QliUR RU8BER CORK ELECTRODE B O f f f R «N«-eT \ SWTEZEJ) C J i f t S S C L A S S fflCKET CHROptffTO&RPiWtC ColSIMN SPECIMEN Wi-ET BUFFER M£T JL f i ' C . 2 2 . £ L t C T R O - C H R O M l \ T O G ; R R P H l C A P P K K M U S (97) APPENDIX (98) PREPPiRMON OF D.E.rVL. CtLLULBSElCOH FOtfn) C E L L U L O S E : as \ DICHLORO DIET/WL 'AMINO ETHYL HYDROCHLORIDE - A/AM OH cxHu | OH H OH H OH DIETHYL AMINO ETHYL CELLULOSE ( S U E C T R C E L OH FOR«V (99) — twee D.E.IVE. CELLULOSE Cji FORM} CO EaUILtBIR&TIOjV -O—T o-ort H of/ OW Ctf, OH D.E.f\L.COXULOSE ( c H 3 c o o , c f FORH) Pw /(• sr (dL) WASHING X } t ^ > -0-0 / / o// O H r-0-OH OH DXJVL CELLULOSE ( a . FORM) + CU3COOH • ( f l f c E T / C flCM>) KPPEHDIX M (100) Bibliography and References 1. ABRAMS, A., KEGELES, G. and HOTTLE, G.A., Journal of Bi o l o g i c a l Chemistry, 1946, Vol. 1964, Page 63. 2. ALEXANDER, P. and BLOCK, R.J., A Laboratory Manual of An a l y t i c a l Methods of Protein Chemistry including Polypeptides, 1960, Vol. 1, Pergamon Press, New York. 3. ALEXANDER, P. and BLOCK, R.J. A Laboratory Manual of An a l y t i c a l Methods of Protein_Chemistry including  Polypeptides, 1961, Vol. II, Pergamon Press, New York. 4. APOSHRIAN, V.H. and KORNBERG, A. Journal of B i o l o g i c a l  Chemistry, 1962, Vol. 237, Page 5lW. 5. BAILEY, Leggett, J. Techniques in Protein Chemistry, 1962, E l s e v i e r Publishing Company, New York. 6. BERGDOLL, M.S., LAVIN, B., SURGALLA, M.J. and DACK, G.M. Science, 1952, Vol. 116, Page 663. 7. BOARDMAN, N.K. Journal of Chromatography, 1959, Vol. 2, Page 389. * 8. BOARDMAN, N.K. and PARTRIDGE, S.M. Biochemical Journal, 1955, Vol. 59, Page 543. 9. BOCK, R.M. and LING, N.S. A n a l y t i c a l Chemistry, 1954, Vol. 26, Page 1543. 10. BOROFF, D.A., RAYNAUD, M. and PREVOT, A.R., Journal of Immunology, 1952, Vol. 68, Page 503. 11. BOWMAN, H.G; and WESTLUND, L.E. , Archives;, of Biochemistry and Biophysics, 1957, Vol. 70, Page 572. ~~ 12. BROWN, F. and CARTWRIGHT, B. Biochemica et Biophysica Acta, 1959, Vol. 33, Page 343. 13. BRUNISH, R. and MOZERSKY, S.N. Journal of B i o l o g i c a l  Chemistry, 1958, Vol. 231, Page 29X 14. BUEHLER, H.J., SCHANTZ, E.J. and LAMANNA, C. Journal  B i o l o g i c a l Chemistry, 1947, Volume 169, Page 2U5~ (101) 15. CALMON, C. and KRESSMAN, T.R.E. Ion Exchangers i n Organic and Biochemistry, 1957, International Science Publications Inc. 16. CARDELLA, M.A., DUFF, J.T., GOTTFRIED, C. and BEGEL, J.S. Journal of Bacteriology, 1958, Vol. 75, Page 360. 17. CARDELLA, A.M., FIOCK, M.A., and WRIGHT, G.G. Ba c t e r i o l o g i c a l Proceedings, 1958, Page 78. 18. CRAWFORD, I.P. and YANOFSKY, C. Proceedings of National  Academy of Science, U.S. 1958, Vol. 44, Page 1161. 19. CREASER, E.H. and TAUSSI, G.A. Virology, 1957, Vol. 4, Page 200. 20. DAVENPORT, F.M. and HORSFALL, F.I. Journal of Experimental  Medicine, 1953, Vol. 91, Page 1450. 21. DOLMAN, C.E. International Congress of Microbiology.  Proceedings 6 th~ Congress, Rome, 1953, Vol. 4, Page 130. 22. DUFF, J.T., WRIGHT, G.G., KLERER, J. and MOORE, D.E., Ba c t e r i o l o g i c a l Proceedings, 1952, Page 107. 23. DUFF, J.T., KLERER, J., BIBLER, R.H., OFTEDAHL, D.M. BEGEL, J.S. and WRIGHT, G.G., Ba c t e r i o l o g i c a l Proceedings. 1954, Page 54. 24. DUFF, J.T., WRIGHT, G.G. and YANOFSKY, A. Journal of  Bacteriology, 1956, Vol. 72, Page 455. 25. DUFF, J.T., KLERER, JULIUS; BIBLER, R.H. ; MOORE, DOROTHY E., GOTTFRIED, C. and WRIGHT, G.G., Journal of Bacteriology, 1957, Vol. 73, Page 597. 26. DUFF, J.T., WRIGHT, G.G., KLERER, J., MOORE, D.E. and BIBLER, R.H. Journal of Bacteriology, 1957, Vol. 73, Page 42. 27. FOX, S.W., and FOSTER, J.F. Introduction to Protein  Chemistry, 1957, John Wiley & Sons, Inc. (102) 28. GAREN, A. and LEVINTHEL, C., Biochemica et Biophysica  Acta, 1960, Vol. 38, Page 470. 29. GENDON, I.Z. Journal of Microbiology. Epidemiology and  Immuno-biology, 1957, Vol. 28, Page 373. 30. GERWING, J., DOLMAN, C.E. and ARNOTT, D.A. Journal of  Bacteriology, 1961, Vol. 81, Page 819. 31. GERWING, J., DOLMAN, C.E. and ARNOTT, D.A. Journal of  Bacteriology, 1962, Vol. 84, Page 302. 32. GORDON, M., FIOCK, M.A., YARINSKY, A. and DUFF, J.T., Journal of Bacteriology, 1957, Vol. 43, Page 533. 33. HEFTMAN, I., Chromatography, 1961, Burgess Publishing Co., Minneapolis, U.S.A. 34. HECKLEY, R.J., HILDEBRAND, G.J. and LAMANNA, C a r l , Journal of Experimental Medicine, 1960, Vol. I l l , Page 745, 35. HIRS, C.H.W., MOORE, S. and STEIN, W.H. Journal of B i o l o g i c a l Chemistry, 1953, Vol. 200, Page 493. 36. KATITCH, R.V., Review of Immunology, 1952, Vol. 16, Page 575. 37. KEGELES, G. Journal of American Chemical Society, 1946, Vol. 68, Page 1 6 7 0 . ~ 38. LAKSHMANAN, T.K. and LIEBERMAN, S. Archives of  Biochemistry and Biophysics, 1954, Vol. 53, Page 259. 39. LAMANNA, C., EKLUND, H.W. and McELROY, O.E. Journal of  Bacteriology, 1946, Vol. 52, Page 1-13. 40. LAMANNA, C a r l and GLASSMAN, H.N. Journal of Bacteriology, 1947, Vol. 54, Page 575. (103) 41. LAMANNA, Carl and DOAK, B.W., Journal of Immunology, 1948, Vol. 59, Page 231. ~ 42.. LAMANNA, C., Proceedings Society Experimental Biology and Medicine, 1948, Vol. 69, Page 332. 43. LAMANNA, C. and LOWENTHAL, J.P. Journal of Bacteriology, 1951, Vol. 61, Page 751. 44. LAMANNA, C. and ARAGON, P.R. B a c t e r i o l o g i c a l Proceedings 1956, Page 94. 45. LEDERER, E., Chromatographic l e chimie Organique et  Biologique, 1960, Vol. 3, Pari s . 46. LEVIN, 0. Archives of Biochemistry and Biophsics, 1958, Vol. 78, Page 33. : 47. LOWENTHAL, J.P. and LAMANNA, C. American Journal of  Hygiene, 1953, Vol. 57, Page 46. 48. MALMSTROM, G. Archives:of Biochemistry and Biophysics 1957, Vol. 70, Page 58. : 49. MARTIN, A.J.P. and PORTER, P.R., Biochemical Journal, 1951, Vol. 49, Page 215. "~ 50. MAXWELL, E.S. and de ROB INCHON, SZULME JSTER, II. Journal of B i o l o g i c a l Chemistry, 1960, Vol. 235, Page 308. 51. NACHOD, F.C., Ion Exchange Theory and Application, 1949, Academic Press Inc., Publishers, New York. 52. NAGEL, W., Charles and VAUGH, Reese H., Archives of Biochemistry and Biophysics, 1963, Vol. 93, Page 344. (104) 53. NEILANDS, G.B., and STUMPF, Paul K. Outlines of Enzyme Chemistry, 1958, John Wiley & Sons, Inc., New York. 54. PAULEUS, S. and NEILANDS, J.B., Acta Chemica Scandinavica, 1950, Vol. 4, Page 1024, 55. PARTRIDGE, S.M., Ion Exchange and i t s Applications, 1955, Society of Chemical Industry, London. 56. PAZUR, J.H. and ANDO, T. Archives of Biochemistry and  Biophysics, 1961, Vol. 93, Page 43. 57. PERUTZ, M.F. Proteins and ^Nucleic Acids, 1962, Els e v i e r , Publishing Company, New York. 58. PETERSON, E.A. and SOBER, H.A. American Chemical Society  Journal, 1956, Vol. 78-1, Page 75TT 59. PETERSON, E.A., WYCKOFF, H.M., and SOBER, N.A. Archives  of Biochemistry and Biophysics, 1961, Vol. 93, Page 43. 60. PORTER, R.R. and PRESS, E.M., Biochemical Journal, 1957, Vol. 66, Page 600. 61. PUTNAM, F.W., LAMANNA, C. and SHARP, D.G., Journal of  B i o l o g i c a l Chemistry, 1946, Vol. 165, Pages 7 3 5 - 7 3 6 . 62. PUTNAM, F.W., LAMANNA, C. and SHARP, D.G. Journal of  B i o l o g i c a l Chemistry, 1948, Vol. 176, Page 401. 63. REITHEL, F.J., Advances i n Protein Chemistry, 1963, Vol. 18, Page l"23~! 64. SAKAGUCHI, G. and TOHYAMA, Y., Japan Journal of Medical  Sciences, and Biology, 1955a, Vol. 8, Page 247. 65. SAKAGjUCHI, G., and TOHYAMA, Y., 1955, Japan Journal of  Medical Sdiences. and Biology, Vol. 8, Page 255. (105) 66. SAKAGUCHI, G. and SAKAGUCHI, S. Journal of Bacterio-logy, 1959, Vol. 78, Page 1. 67. SAKAGUCHI, G., and SAKAGUCHI, S. , and HISASHI, K., Japanese Journal of Medical Science and Biology, 1963. Vol. 16, No. 5, Page 309. 68. SCHACHMAN, H.K. Ultracentrifugation, D i f f u s i o n and Viscometers, Reprint Methods i n Engymplogy, l o l . IV, 1957, Academic Press Inc., Publishers, New York. 69, SCHWIMMER, S. and PARDEE, A.B., Advances i n Engymology, 1953, Vol. XIV, Page 375. 70. SHAINOFF, J.R., and LAUFFER, M.A., Archives of Biochemistry and Biophysics, 1956, Vol. 64, Page 415. 71. SHEPARD, C C . and TISELIUS, A. Discussions Faraday  Society, 1949, Vol. 7, Page 275. 72. SHUKUYA, R. and SCHWERT, G.W., Journal of B i o l o g i c a l  Chemistry, 1960, Vol. 235, Page 1649. 73. SIMONART, P. and CHOW, K.Y. Enzymologia, 1951, Vol. 14 Page 356. 74. SNIPE, P.T. and SOMMER, H., Journal of Infectious Diseases, 1928, Vol. 43, Page 152. 75. SNOSWELL, A.M. Biochemica et Biophysica Acta, 1959, Vol. 35 Page 574. 76. SOBER, H.A., GUTTER, F.J. WYCKOFF, M.M. and PETERSON, E.A., Journal of the American Chemical Society, 1956, Vol. 78, Page 756. 77. SOMMER, E.W., SOMMER, H. and MEYER, K.F. Journal of  Infectious Diseases 1926, Vol. 39, Page 345-50. 78. SOMMER, H. and SNIPE, P.T. Journal of Infectious Diseases 1928, Vol. 43, Page 145. (106) 79. SOMMSR, H., NEALSON, P.J. and SNIPE, P.T. Journal  of Infectious Diseases, 1928, Vol. 43, Page 161. 80. SOMMER, E.W., and SOMMER, H. Journal of Infectious  Diseases 1928, Vol. 43, Page 497. 81. SOMMER, H. Proceedings Society Experimental Medicine and Biology, 1936, Vol. 35, Page 520. 82. STERNE, M. and WENTZEL, L.J. Journal of Immunology, 1950, Vol. 65, Page 175. 83. STOCKINGER, H.E. and ACKERMAN, H. Journal of Bacterio-logy, 1941, Vol. 42, Page 136. 84. SWINGLE, S.M. and TISELIUS, A. Biochemical Journal, 1951, Vol. 48,Page 171. 85. TISELIUS, A. Biochemical Journal, 1951, Vol. 48, Page 171. 86. TISELIUS, A., HJERTEN, S. and LEVIN, O., Archives .of:,: Biochemistry and Biophysics, 1956, Vol. 65, Page 132. 87. TISELIUS, A. and HAGDAHL, L. Acta Chemica Scandinavica, 1950, Vol. 4, Page 394. : 88. TRISTRAM, G.R. and SMITH, R.H. Advances i n Protein  Chemistry, 1963, Vol. 18, Page "527" 89. TURBA, F. Advances i n Enzymology, 1960, Vol. 22, Page 417. 90. VINET, G. and FREDETTE, V., Science, 1951, Vol. 114, Page 549. 91. WAGMAN, J. and BATEMAN, J.B. Archives of Biochemistry and  Biophysics, 1951, Vol. 31, Page 424. (107) 92. WAGMAN, J. and BATEMAN, J.B. Archives of Biochemistry  and BIOPHYSICS, 1953, Vol. 45, Page 375. ~ 93. WAGMAN, Jack. Archives of Biochemistry and Biophysics, 1954, Vol. 50, Page 104. 94. WENTZEL, L.M., STERNE, M. and POLSON, A. Nature 1950, Vol. 166, Page 739. 95. ZECHMEISTER, L., TOTH, G. and BALINT, M. Enzymologia, 1938, Vol. 5, Page 302, 96. ZIMMERMAN, S.B., K0RNBERG, S.R. and KORNBERG, A. Journal of B i o l o g i c a l Chemistry, 1962, Vol. 237, Page 512. 97. ZITTLE, Charles A. Advances i n Enzymology, 1953, Vol. 14, Page 319. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0104867/manifest

Comment

Related Items