]~Ied. Microbiol. Immunol. 159, 89--100 (1973) 9 by Springer-Verlag 1973

Discrimination between Binding to CNS, Toxicity and Immunreactivity of Derivatives of Tetanus Toxin E. H a b e r m a n n Pharmakologisches Institut der Justus Liebig-Universit~t GieBen Received August 3, 1973 Abstract. 12~I-tetanus toxin was modified by oxidation with chloramine T, iodination, acetylation, and treatment with formol. The following changes were studied: Binding to brain homogenate and to solid phase antibody in vitro; production of generalized tetanus and binding to whole spinal cord in situ (mice); production of local tetanus and binding to the lumbosacral cord in situ (rats). 1. There is no strict correlation between antigenic reactivity and binding to brain homogenate in vitro. For instance, massive formol treatment leaves the binding to solid phase antibody nearly intact but nearly abolishes the latter process. 2. There is no strict correlation between the binding to brain homogenate in vitro and binding to spinal cord in vivo, as shown with acetylated toxin. The inability of this modified toxin to react in vivo is accompanied by (and probably due to) its fast decay from blood plasma. 3. There is no strict correlation between the binding of modified toxins to spinal cord in rats (model of local tetanus) or mice (model of generalized tetanus) and their toxicities. For instance, mild treatment with formol partially preserves the in vivo affinity to spinal cord whereas the toxicity decreases to a nonmeasurable range. It is concluded that fixation to spinal cord in vivo is a prerequisite of the pharmacological activity of tetanus toxin. However, fixation by itself, is insufficient to elicit the action of the toxin.

T e t a n u s t o x i n occupies a u n i q u e p o s i t i o n a m o n g t h e n e u r o t o x i n s acting a t t h e CNS level, since i t is fixed to i t s t a r g e t organ. B r a i n m a t t e r ( W a s s e r m a n n a n d T a k a k i , 1898), s y n a p t o s o m e s p r e p a r e d f r o m b r a i n (Mellanby et al., 1964), a n d c o m p l e x e s b e t w e e n gangliosides a n d eerebrosides (see v a n H e y n i n g e n a n d M e l l a n b y , 1973) are a b l e to r e m o v e the t o x i n f r o m i t s solution. T h e s t u d y o f such processes has been facilit a t e d b y using l a b e l e d t o x i n ( H a b e r m a n n , 1972, 1973; H a b e r m a n n a n d Dimpfel, 1973; H a b e r m a n n et al., 1973 ; W e l l h 6 n e r et al., 1973 a, b ; t t e n sel et al., 1973; Seib et al., 1973). T h e l a b e l has b e e n localized in v i v o b y a u t o r a d i o g r a p h y ( H a b e r m a n n et al., 1973) a n d h i s t o a u t o r a d i o g r a p h y (Dimpfel a n d H a b e r m a n n , u n p u b l i s h e d ) . H a b e r m a n n et al. (1973) h a v e shown t h a t t h e l a b e l e d m a t e r i a l deposited in t h e tissues is i n d i s t i n g u i s h a b l e f r o m t e t a n u s t o x i n b y v a r i o u s

90

E. Habermann

techniques. However, the o b j e c t i o n c a n n o t be ruled o u t t h a t the label is c o n n e c t e d i n fact n o t with n a t i v e t o x i n b u t w i t h d e r i v a t i v e s toxoided d u r i n g the p u r i f i c a t i o n (Pillemer a n d Moore, 1948), or d u r i n g the labeling, especially b y the o x i d a t i o n i n v o l v e d ( H a b e r m a n n , 1972), or in rive. The a m o u n t s of t o x i n to be expected i n the CNS of poisoned a n i m a l s are too small to be characterized except b y radiological procedures. P r e v i o u s l y I h a v e observed ( H a b e r m a n n , 1973) t h a t t e t a n u s toxoid labeled with 125I is, a l t h o u g h v e r y weakly, adsorbed to washed brain h o m o g e n a t e . This was a n i m p o r t a n t evidence for a possible dissociation of n e u r o t r o p y , t o x i c i t y a n d i m m u n o r e a c t i v i t y . I t p r o m p t e d us to prepare chemically modified radio-labeled toxins a n d to compare t h e alterations of the three properties m e n t i o n e d . Material and Methods 1. Reagents a) Tetanus toxin, purified according to Bizzini et al. (1969), was a gift o. Dr. Bizzini and Dr. Turpin, Institut Pasteur, Paris. Originally, its LD 50 (mouse s. c.), was 2.5--3.3 ng/kg. During storage, it deteriorated by approximately 500/0. The toxin was labeled according to Habermann (1972) using 8 tzg ehloramine T per sample. Its initial specific radioactivity was 7 mCi/mg and its toxicity had decreased to 10 ng/kg (mouse, s. c.). The labeled toxin (final concentration 1:60000) was stored at ~ 4 ~ in the presence of 10~ rabbit serum, 0.005 M Na~ EDTA, 0.025 M phosphate buffer pit 7.5, and half-saturated ammonium sulphate. Attempts to work with labeled toxin in the absence of stabiIizing agents led to uncontrollable losses and had to be abandoned. Immediately before use, the 12~I-toxin was diluted tenfold with water and dialyzed first against phosphate-buffered saline (PBS) (0.05 1~ phosphate pH 7.5) containing 0.1 ~ sodium iodide and 0.1 ~ Na2S20~, then against two changes of phosphate-buffered saline without additives. By that, the amount of radioactivity not precipitable with trichloroacetic acid was kept below 5 ~ The immunoreactivity of the protein was, depending on the sample, about 840/0. About 230/0 of the iodinated material reacted with 50/0 brain homogenate in vitro; this is, however, a minimum value, since higher concentrations of the homogenate are able to bind more toxin (see Habermann, 1973). b) Preparation of the derivatives. We checked the behaviour of the labeled toxin against iodination (coupled with oxidation), oxidation with chloramine T, acetylation, and treatment with formol. One out of about 10 experiments was done as follows. Iodination. 0.3 ml of dialyzed tetanus toxin (dilution 1/10 in PBS; proteinbound iodine: 960/0 of total; 2.9 [zCi/ml of 1.75 mCi/mg toxin) was mixed with the same volume of sodium iodide (either 10-3 or 10-8 mg/ml in PBS). Chloramine T (0.1 mg/ml; 0.3 ml) was added and its excess destroyed 1 min later with 30091 of Na~S205 (0.1 mg/ml). The mixture was made up to 1.5 ml with PBS containing 5 mg/ml bovine serum albumin (Behringwerke, dried, purified). Oxidation. The same mixture was used as for iodination; however, the iodide solution was replaced by PBS. In order to modify the protein still further, a solution containing i mg/ml chloramine T was applied to another incubation mixture. Formol Treatment. The diluted 125I-toxin solution (300~1) was incubated overnight at room temperature with the same volume of formol (Merck, p.a.)

Binding of Modified Tetanus Toxins

91

diluted with PBS to 1 ~ and 0.1 ~ (W/V), respectively. Then the incubates were made up to 1.5 ml with PBS containing 0.50/0 albumin. Acetylation. The diluted 12~I-toxin solution (0.3 ml) was mixed with the same volume of a saturated solution of sodium acetate, cooled to 0 ~ C, incubated with 3 [xl of acetic anhydride for 1 h, and made up to 1.5 ml with PBS containing 5 mg/ml serum albumin. A control was always run simultaneously, consisting of 300 [zl of PBS-diluted (1/10) and dialyzed toxin, 600 bd of PBS, 300 lzl of Na2S2Oa solution (0.1 ~ ), and 300 [zl of PBS containing albumin. The concentration of sodium metabisulphite used does not influence the toxicity. PBS containing 0.5 ~ albumin was used as diluent, if not otherwise indicated. c) Binding agents. Whole rat brain was homogenized with 10 volumes of water in an Uitra-Turrax (Janke and Kunkel, Staufen/Breisgau, Germany) and lyophilized. Before use, weighed samples were washed three times (Eppendorf-Zentrifuge) with PBS containing 0.5~ albumin, and reconstituted with the same solution to the desired concentration. Since the fixation of toxin to brain does not follow saturation characteristics, we arbitrarily chose homogenate concentrations of 5 ~ and 0.5 ~ (w/v).--Liver homogenate from rats was prepared in the same manner. -The coupling of bromoacetyl cellulose with equine antitetanus serum (Fermo, Behringwerke, 3000 U/ml) was done according to Robbins et al. (1967) as described previously (Habermann, 1970). That amount of solid phase antitoxin was determined in pilot experiments which was more than enough to fix the immunreaetive moiety of the labeled toxin preparation applied.

2. Binding Assay8 All tests were done as triplicates in Eppendorf vessels. Aliquots of the incubation mixtures (see 1. Reagents) were diluted 100 fold with PBS containing 0.5 ~ albumin. In order to avoid non-specific adsorption to surfaces, a small amount of unlabeled toxin (0.2 izg/ml) was incorporated into the diluant. Controls showed that the binding assays were not altered by this ingredient. The diluted samples (0.1 ml) were mixed with 0.2 ml of washed brain homogenate, liver homogenate, solid phase immunosorbent, and diluant (PBS with 0.50/o albumin), respectively. The Eppendoff vessels were rotated for 2 h at room temperature around their horizontal axis and centrifuged (Eppendorf centrifuge, about 8000)< g). 0.2 ml of the supernatant (= a) and the remaining contents (~) of the vessels were counted (Autogamma spectrometer, Packard) for their radioactivities. The radioactivity bound to the sediment ( ~ b) was calculated (Olivetti Programma) according to the formula

b=~--O.5a. 3. Animal Experiment8 Toxicity tests were made in N M R I mice of both sexes, 15--25 g, own b r e e d . The potency of the modified toxins to elicit local tetanus was assessed by injecting 0.2 ml/100 g body weight of the undiluted or 1/a diluted incubation mixtures into the right m. gastrocnemius of rats (Wistar, about 200 g, both sexes). The spinal cord of the poisoned rats was dissected and counted as described previously (Itabermann, 1972).--Generalized tetanus was studied in mice injected intravenously with serial (1:1) dilutions of the various incubation mixtures. After 50 h paired animals were killed by exsanguination in slight ether anesthesia. Blood was collected for counting 0.05 ml of serum before and after precipitation with triehloroacetic acid. The spinal cords were dissected out immediately after sacrifice, weighed in a closed Eppendorf vessel, and counted for their radioactivities.

92

E. H a b e r m a n n

All animals had ad libitum access to drinking water containing 0.5 a/0 sodium iodide, and to Altromin~ pellets. 4. Signi/icance of the Results E a c h set of experiments (ir~ vitro; in vivo with rats; in vivo with mice) was done several times with different batches of modified toxins. For the sake of exact comparison, all t h e modified toxins were compared with t h e control b y injecting the animals a t the same day. Although the n u m b e r of animals t h a t could be handled simultaneously was restricted, we t h i n k the significance of the animal experiments

to be sufficient because a) controls, done in triplicate, had a satisfactory standard deviation, b) all experiments were done with graded doses, c) all experiments were repeated several times, d) corresponding results were obtained with mice and rats.--The in vitro experiments were also repeated several times. The means, their standard deviations, and the p values were calculated (Student's t test) from one experiment (Table 1) done at 1 day in triplicate. Results 1. Binding in vitro As expected, the ability of tetanus toxin to react with solid phase antitoxin is only slightly affected by iodination, acetylation, or by treatment with formaldehyde. Oxidation is ineffective in this respect when performed with the lower amount of chloramine T, whereas the higher dose of the oxidant considerably impairs the antigen-antibody intera c t i o n . - - T h e interaction with brain homogenate is much more sensitive. Except acetylation, every other procedure affects it more or less.--For comparison, experiments with liver homogenate were enclosed. Since a specific binding of toxin to liver homogenate does not take place, these values represent non-specific interactions (Table 1). The findings indicate t h a t very different modifications of the toxin molecule affect in the first line the sites responsible for its interaction with CNS m a t t e r whereas the antigenic reactivity is relatively resistant. 2. Binding in vivo Our investigations were undertaken in order to give evidence for or against an intimate correlation between fixation and action of tetanus toxin. I n vitro studies alone would have been inconclusive, since the modifications of the toxin could differ not only b y their affinity towards CNS m a t t e r but also b y their pharmacokinetic behaviour. Therefore we tested the substances for their ability to elicit generalized tetanus in mice and local tetanus in rats, and measured the radioactivities of the spinal cords in parallel assays. a) Generalized Tetanus in Mice T r e a t m e n t of the labeled toxin with the higher concentration of chloramine T nearly abolishes the toxicity. Binding to spinal cord still

77.9 4- 1.31

70.2 4- 2.02** 76.4 4- 4.11

53.4 4- 1.07"** 78.9 4- 1.56

73.9 4- 2.62

69.6 :t: 1.27 74.1 ~: 0.42

Control

Sodium iodide 1:105 (with 1:10 * chloramine T)

Chloramine T 1:10 a 1:10 ~

Acetylation

Formoi 1:10 z 1:103 1.7 4- 6.94** 6.2 4- 0.55***

21.5 4- 3.05

5.5 4- 1.73"** 18.8 • 1.33

10.0 4- 0.99** 14.7 • 3.62

20.5 4- 2.15

B r a i n homogenate (5 ~

1.1 -]- 1.18"** 3.5 :J: 0.31"*

12.1 :t: 0.29

5.7 :[: 0.27** 9.5

3.7 :[: 1.42"* 6.8 4- 3.72*

14.3 4- 2.16

Brain homogenate (0.5 ~ )

9 P > 0.01 < 0.05; ** P > 0.001 < 0.01; *** P < 0.001 (against the control values).

Solid-phase antitoxin

Sample

--1.1 4- 0.85*** --0.7 :[: 0.75***

7.7 4- 1.57

5.8 4- 3.18 6.4 -V 5.39

8.8 :t: 3.03 6.3 4- 2.25

6.4 4- 0.29

Liver homogenate

Table 1. Interaction of chemically modified, labeled toxin with homogenates a n d solid-phase antibody For the incubation mixtures a n d the calculations see "Methods". About 800 cpm was added per vessel. The quantities of b o u n d radioactivity are given as percent 4- S. D.

e~

O ~4

O

0~

Table 2. Toxicity, e n r i c h m e n t in t h e spinal cord a n d s e r u m c o n c e n t r a t i o n s of radioactive m a t e r i a l in mice injected w i t h modified labeled t e t a n u s t o x i n T h e control v a l u e s r e p r e s e n t & i Sample

~;

Radioactivity in t h e spinal cord (cpm/g wwb)

R a d i o a c t i v i t y in blood serum(cpm/50txlb) Total

TCApreeipitable

6-20; 6-20 20-24; 20-24 28-42 (6 animals) 42; 52; 52-68 52-70; 52-70; 70-78 > 100; > 100; 80-112

---340 • 18 150 ~: 9 74 ~ 5

---27.1 :[: 2.6 8.0 • 1.4 4.1 + 1.1

---29.6 • 6.6 9.3 • 1.6 4.9 • 1.9

S o d i u m iodide 150 1:105 75 (with 37.5 e h l o r a m i n e T ) 19

48; > 1 0 0 ; >100 >100 >100

145 53 43 13

50 20 10 6

51 21 10 4

Chloramine T 1 : 10 a

150 75 37.5 19 9.5

80-100 c >100 >100 > 100 >100

291 220 135 86 26

27 16 7 2 3

30 18 7 3 3

Chloramine T 1:104

150 75 37.5 19 9.5 4.7

6 - 2 0 ; 6-20 20-24; 20-24 32-44; 32-44 47 80-100 > 100

---389 184 --

---15 4 --

---15 6 --

Aeetylation

300 150

> 100 > 100

-9

-4

-1

Formol 1 : 102

300 150 75

> i00 > 100 > 100

-64 33

-27 13

-26 12

Formol I : 10 a

300 150 75 37.5 19

> > > > >

-1186 638 314 33

-62 42 23 2

60 44 24 2

Control

Toxin concentration~ injected (ng/ml)

Death time (hours)

150 75 37.5

n = 3

I9

9.5 4.7

100 100 100 100 100

>100

--

10 [zl/g a n i m a l injected i.v., c o r r e s p o n d i n g to a b o u t 4500 cpm, of t h e solution containing 150 n g t o x i n / m l . b F r o m a n i m a l s killed 50 h a f t e r injection. T h e w e i g h t of t h e spinal cords was, d e p e n d i n g o n t h e b o d y w e i g h t a n d k i n d of dissection, b e t w e e n 66 a n d 81 rag. c D e a t h w i t h o u t t y p i c a l s y m p t o m s of t e t a n u s . -- = n o t tested.

Binding of l~Iodified Tetanus Toxins

95

occurs, but it is diminished when compared with mice having received the same amounts of control toxin. The serum radioactivity obtained with the more strongly oxidized sample is lower t h a n that in the controls. Therefore both decreased affinity (see Table 1) and altered pharmacokinetics might have contributed to the reduction of toxicity and of binding to spinal cord in vivo. The lower concentration of chloramine T does affect neither the toxicity nor the binding to spinal cord as compared with the controls. The same is true for toxin treated with iodine. Toxicity is nearly abolished, and so is the fixation to spinal cord in vivo. Acetylation abolishes the toxicity to a non-measurable degree (less than 1/32 of the control). The radioactivity of the spinal cord and in the blood serum was at the limit of detection. We m a y conclude t h a t acetylatcd toxin, although fixed to brain m a t t e r in vitro (see Table 1), cannot interact with spinal cord in vivo because it is eliminated too fast. Formol Treatment. As expected, both concentrations of formol annul the toxicity. The higher concentration {10 -2 g]ml) also reduces the radioactivities in both serum and spinal cord considerably. This is in agreement with our previous findings (Habermann, 1972; H a b e r m a n n and Dimpfel, 1973), and also with the inability of this derivative to react with brain m a t t e r in vitro (see Table 1}. Quite unexpected, however, was the behaviour of the toxin treated with the lower {10-3 g/ml) concentration of formol. With this preparation, very high radioactivities in both serum and spinal cord can be achieved in vivo without a n y signs of toxicity. When calculated from the radioactivity injected, the percentage bound to spinal cord in vivo (Table 2) and also the serum radioactivity is lower t h a n in the controls. Due to the lack of toxicity, however, much (at least 32 times) more of the labeled, formol-treated toxin can be injected, raising both serum and spinal radioactivity to levels not attainable with control toxin. Thus a clearcut dissociation between toxicity and binding to spinal cord in vlvo is to be noted with that modified toxin. Any modification of the toxin diminishes the serum concentration achieved with a given concentration injected. However, there is no close parallelism between the reduction of the spinal and of the serum radioactivities. For instance, t r e a t m e n t with iodine diminishes the serum radioactivity to a lesser degree and the spinal radioactivity to a higher degree when compared with the higher concentration of chloramine T. Thus the diminution of the spinal radioactivity m a y be partially, but not totally due to the reduced radioactivity in the serum. Whereas the toxicities of the other modifications might depend to same degree on the serum radioactivity, this is clearly not the case with the modification

96

E. ttabermann

prepared with low concentrations of formol. The animals remain sympt o m l e s s a l t h o u g h theh" s e r u m r a d i o a c t i v i t y is 7 - - 8 t i m e s h i g h e r t h a n in the controls poisoned to death.

b) Local Tetanus in Rats A c c o r d i n g to o u r v i e w , g e n e r a l i z e d t e t a n u s is n o t h i n g else t h a n a m u l t i p l e l o c a l t e t a n u s ( H a b e r m a n n a n d D i m p f e l , 1973). N e v e r t h e l e s s we s t u d i e d t h e m o d i f i e d t o x i n s b y a i d o f t h i s m o d e l , e s p e c i a l l y b e c a u s e the p h a r m a c o k i n e t i c p e c u l i a r i t i e s m i g h t d e p e n d o n t h e m o d e o f t o x i n ap-

Table 3. Local tetanus and radioactivity in the lumbosacral part of the spinal cord of rats. 0.9. ml/100 g body weight of the variously modified toxins were injected into the right m. gastroenemius. The animals were killed 48 h later Sample

Toxin coneentration injected (ng/ml)

Severity of symptoms

Lumbosacral radioactivity (total epm)

Control (~ -4- S. d., n = 3)

300 150 75 38

+++ +++ +++ ++

234 112 92 36

Sodium iodide 1 : 105 (with chloramine T)

300 60

0

Sodium iodide 1 : 10n (with chloramine T)

300 60

+++ +

229 42

Chloramine T 1 : 10 ~

300 60

0 0

13 -- 1

Chloramine T 1 : 106

300 60

+++

296

Acetylation

300 60

0 0

-- 1 0

Formol 1 : 102

300 60

0 0

18 0

Formol 1 : 10 a

300 150 60

0 0 0

237 72 38

+++ ++ + 0

(+)

+ +

• 67 • 10 • 7 • 13

19 3

59

= s e v e r e local tetanus (rigidity exceeding the injected limb). = strong local tetanus (rigidity r e s t r i c t e d t o t h e injected limb). : r i g i d i t y w e l l visible. ~ no rigidity.

Binding of Modified Tetanus Toxins

97

plication and on the animal species used. Rats were injected either with undiluted or 115 diluted solutions of the modified toxins (see "Methods") and exsanguinated in slight ether anesthesia about 48 h later. If there was any em'ichment of radioactivity, it was always found in the lumbosacral part of the spinal cord. For the sake of simplicity, the combined radioactivity of these spinal segments was contrasted with the severity of symptoms (Table 3). The results with local tetanus in rats are not different from those with the generalized tetanus in mice. The higher concentration of the oxidant destroyed both toxicity and binding to the spinal cord, whereas the lower concentration left the toxin practically unaltered. The higher concentration of iodine inactivated the toxin in both respects nearIy completely. Aeetylation abolished both toxicity and binding. Again, the lower concentration of formol rendered the material nontoxic, but kept its neurotropy well preserved. Discussion

a) No correlation between the immunoreactivity of tetanus toxin and its interaction with CNS matter in vivo and in vitro. It is amply known that treatment with formol converts tetanus toxin into its toxoid. Toxoid is still able to react with CNS matter in vitro, since it partially prevents the fixation of toxin (Wolters and l~ischoeder, 1954). A competition between toxoid and toxin can be deduced from the ability of high doses of toxoid to protect animals instantly against otherwise lethal toxin doses (for review see Prdvot, 1955). After intramuscular (Habermann, 1972) or intravenous (Habermann and Dimpfel, 1973) injection of l~sI-toxoid in rats, radioactivity can be found in the spinal cord. Admittedly much higher amounts of labeled toxoid than toxin have to be app]ied in order to achieve a measurable fixation to the CI~S. l~SI-toxoid also interacts with brain homogenate, although weakly, which can be prevented by cold toxin (Habermann, 1973).--0ur present experiments give direct evidence that the formol concentrations used leave the immunorcactivity nearly intact whereas the fixation to brain homogenate is decreased to a just (formol 10 -a) or not (formol 10 -2) measurable range. Similarly, the higher concentration of chloramine only partially prevented the fixation to solid-phase antibody, whereas the interaction with brain homogenate was reduced to a non-measurable degree. Correspondingly, some immunoreactive derivatives of the toxin may have lost totally or partially the ability to become fixed to spinal cord in vivo (for compilation see Table 4). Immunoreaetive sites can be clearly distinguished from sites binding to CNS matter, as has been already done by others (see Kryzhanovsky, 1973). 7 i~Ied.5Iicrobiol. Immuno]., Bd. 159

E. Habermann

98

Table 4. Compilation of the properties of the modifications of toxin in vitro

Control Strong iodination Weak iodination Strong oxidation Weak oxidation Acetylation Strong formol treatment Weak formol treatment

in viw

Binding to solid-phase antibody

Binding to brain homogenate

Toxicity

Binding to spinal cord

+++ ++ +++ + (+) +++ +++

+++ + ++ 0 +++ +++

+++ (+) +++ (+) +++ 0

+++ (+) +++ + +++ 0

++

0

0

(+)

+++

+

0

++

b) N o s t r i c t c o r r e l a t i o n b e t w e e n b i n d i n g to spinal cord i n vivo and to b r a i n m a t t e r in vitro. I t c a n be p r e s u m e d t h a t t h e i n vivo a n d t h e in vitro i n t e r a c t i o n s b e t w e e n t e t a n u s t o x i n a n d CNS m a t t e r a r e g o v e r n e d b y t h e s a m e chemical forces (see H a b e r m a n n , 1973). T h e m o r e a s t o n i s h i n g was t h e fact t h a t a e e t y l a t e d t o x i n was well b o u n d i n vitro b u t n o t in vivo. This e x c e p t i o n a l b e h a v i o u r m u s t h a v e been due t o a p h a r m a c o k i n e t i c pecul i a r i t y . I n d e e d t h e a c e t y l a t e d t o x i n h a d d i s a p p e a r e d f r o m t h e blood p l a s m a 48 h a f t e r i t s i n j e c t i o n in mice. T h u s i t c a n be a s s u m e d t h a t its r a p i d clearance is a t l e a s t one cause o f its failure to b i n d in vivo. To a lesser degree, a n e n h a n c e d e l i m i n a t i o n from t h e p l a s m a m i g h t also c o n t r i b u t e to t h e r e d u c e d spinal a c c u m u l a t i o n of t h e o t h e r t o x i n derivatives. e) N o s t r i c t c o r r e l a t i o n b e t w e e n t h e b i n d i n g to spinal cord in vivo a n d t h e p r o d u c t i o n of local or g e n e r a l t e t a n u s . T a b l e 4 shows t h a t t h e c o r r e l a t i o n b e t w e e n b i n d i n g to spinal cord in r a t s or mice, a n d t h e a b i l i t y to elicit local or s y s t e m i c t e t a n u s is generally good. This is i m p o r t a n t w i t h r e s p e c t to t h e i n t e r p r e t a t i o n of our p r e v i o u s r e s u l t s b a s e d on t h e use o f - - a d m i t t e d l y m u c h m o r e c a u t i o u s l y - - o x i d i z e d a n d i o d i n a t e d t o x i n . H o w e v e r , i t c a n n o t be e x c l u d e d t h a t originally i n t a c t t o x i n is t o t a l l y or p a r t i a l l y c o n v e r t e d in t h e b o d y to a d e r i v a t i v e which becomes (or r e m a i n s ) fixed to i t s t a r g e t o r g a n w i t h o u t m a i n t a i n i n g a visible t e t a n u s . This w o u l d be one, b u t n o t the only, e x p l a n a t i o n w h y

Binding of Modified Tetanus Toxins

99

r a d i o a c t i v i t y in t h e spinal cord can o u t l a s t t h e d u r a t i o n o f s y m p t o m s ( H a b e r m a n n a n d D i m p f e l , 1973). The d i s s o c i a t i o n b e t w e e n t h e in vivo b i n d i n g a n d t h e t o x i c i t y is, however, s t r i k i n g w i t h t h e t o x i n s u b j e c t e d to w e a k formol t r e a t m e n t (see T a b l e 2 a n d 3). This e x p e r i m e n t clearly i n d i c a t e s t h a t fixation a n d action o f t e t a n u s t o x i n are t o be d i s t i n g u i s h e d . Conversely, no modified toxin h a s been f o u n d which w o u l d a c t w i t h o u t being fixed to spinal cord in vivo. T h u s t h e conclusion is o b v i o u s t h a t t h e fixation o f t h e toxin is a p r e r e q u i s i t e of its action. W h y t h e n is t h e m a t e r i a l t r e a t e d with low c o n c e n t r a t i o n s of formol p r a c t i c a l l y n o n - t o x i c a l t h o u g h being bound b y spinal cord in vivo ? F i r s t l y , we could a s s u m e a t l e a s t one binding a n d a t l e a s t one a d d i t i o n a l " t o x i c " site a t the surface of t h e toxin molecule. The f o r m e r s h o u l d be m u c h m o r e r e s i s t a n t to formol than t h e l a t t e r . Secondly, t h e t e r t i a r y s t r u c t u r e of t h e t o x i n molecule could be a l t e r e d b y formol so t h a t t h e h y p o t h e t i c a l t o x i c site, a l t h o u g h u n d a m a g e d , m i g h t be u n a b l e to r e a c h its " r e c e p t o r " a t t h e cell. T h i r d l y , the f o r m a l i n i z e d " t o x i n " m i g h t be fixed to a n y silent r e c e p t o r b u t n o t to the a c t i v e r e c e p t o r m e d i a t i n g t h e t e t a n u s . Because none of these a l t e r n a t i v e s can be r u l e d out, i t w o u l d be p r e m a t u r e to t a k e t h e existence of different toxic a n d b i n d i n g sites a t t h e t o x i n molecule as e s t a b l i s h e d . Others (see K r y z h a n o v s k y , 1973) d r a w t h e i r conclusions in f a v o r o f t h e existence of d i s t i n c t b i n d i n g a n d t o x i c groups f r o m t h e c i t e d experiments w i t h t o x o i d . Our c a u t i o u s r e m a r k s a p p l y to t h e m too.

Acknowled!lement.~. Dr. R~ker labeled the tetanus toxin. Mrs. Irmtraud Heller gave deliberate and skilful technical assistance. Dr. Bizzini and Dr. Turpin subsidized us with purified toxins. I am indebted to all them.

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