Studies on the status of arginine residues in cobrotoxin

Biochimica et Biophysica Acta, 365 (1974) 1-14

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36816 STUDIES ON T H E STATUS OF A R G I N I N E RESIDUES IN COBROTOXIN ~

C. C. YANG a, C. C. CHANG b and I. F. LIOU a •lnstitute of Molecular Biology, National Tsing Hua University, Hsinchu, and bDepartment of Biochemistry, Kaohsiung Medical College, Kaohsiung, (Taiwan)

(Received April 5th, 1974)

SUMMARY Reaction of cobrotoxin with phenylglyoxal at pH 8.0, resulted in almost complete loss of lethal activity and four of the six arginine residues were modified consequently. However, the rate of inactivation was decreased significantly when the pH of the reaction was lowered. Only one arginine residue at position 28 was modified at pH 6.0 and the product retained full biological activity. Arg-33 is the next one modified when the reaction was carried out at pH 6.7 and the lethality drops precipitously, but the antigenic activity was not altered significantly. However, the lethal activity was lost almost copmletely and the antigenic activity decreased about 30 7o when an additional arginine residue at position 30 was modified at pH 7.5. These results indicate that Arg-30 and Arg-33 are functionally important for the lethal activity and Arg-30 and Arg-36 are more closely related to the antigenic specificity of the toxin.

INTRODUCTION Cobrotoxin, a neurotoxic crystalline protein, was isolated from the venom of Taiwan cobra (Naja naja atra) [1] and was proved to be the main toxic protein in cobra venom [2]. The two-dimensional structure of the toxin which has recently been established [3, 4] permits a study of structure-function relationships. Previous studies [5-9] on the chemical modification of the single tryptophanyl, tyrosyl and histidyl residues, free amino and carboxyl groups in cobrotoxin suggested that either the intact Trp-29, Tyr-25, His-32, e-amino group of Lys-47 or y-carboxyl group of Glu-21 is essential for full activity of the toxin. Cobrotoxin is a basic protein with six arginine residues at the positions 28, 30, 33, 36, 39 and 59 in the sequence (Fig. 1). After modification with 1,2-cyclohexanedione [10, 11, 12] in strong alkaline solution (0.2 M NaOH), all arginine residues were modified and lethal activity lost completely. If the reaction was conducted in 0.1 M triethylamine buffer (pH 10), four residues were modified and lethality decreased to 1.6 ~o (unpublished observations). The reaction conditions used, however, were not * A part of this paper was presented at the 9th International Congress of Biochemistry at Stockholm, 6th July, 1973.

,~--

24

30 39 36

STRUCTURE OF COBROTOXIN

r~~$~,~-.~ v59 .~ ~

v~}~_.O ~

H

Fig. 1. Structure of cobrotoxin. Two-dimensional schematic diagram showing the arrangement of the disulfide bonds and sequence of the amino acid residues. mild enough for the biologically active protein and the reagent appears to react with e-amino group of lysine residue simultaneously. Recently Takahashi [13] has reported that a group specific reagent, phenylglyoxal, can react highly specifically with guanidino group of arginine residue in protein under mild conditions. In the present study, selective and stepwise chemical modification of arginine residues were conducted with phenylglyoxal and the degree of modification in relation to the lethal activity and antigenic specificity have been studied ill details. From the results of this investigation, the arginine residues which are important for the biological functions of cobrotoxin have been differentiated, and the positions of these residues in the amino acid sequence have also been established. MATERIALS AND METHODS Cobrotoxin used in this study was prepared from Taiwan cobra (Naja naja atra) venom as previously described [1] and the homogeneity was verified by acrylamide gel electrophoresis. Phenylglyoxal hydrate was purchased from Seikagaku Kogyo Co., Ltd. and N-ethylmorpholine from Nakarai Chemicals, Ltd. Trypsin and chymotrypsin were from Worthington Biochemical Corp. Reagent grade/3-mercaptoethanol and iodoacetic acid obtained from Matheson and Coleman Co. were used. Sephadex G-25 and CM-cellulose were purchased from the Sigma Chemical Co. and urea was a Mallinckrodt reagent. All other reagents were of analytical grade.

Reaction of phenylglyoxal with cobrotoxin Modification of arginine residues in cobrotoxin with phenylglyoxal was per-

formed essentially according to the method described by Takahashi [13]. To a solution of cobrotoxin (4 #moles) in 0.5 ml of 0.2 M N-ethylmorpholine acetate buffer (pH 8.0), a 100-fold molar excess of phenylglyoxal in 1.5 ml of the same buffer was added, and the reaction was allowed to proceed at room temperature (27 °C) for 1 h. Reactions at pH values other than 8.0 were carried out in the same way with appropriate buffers. The mixture was passed through a column of Sephadex G-25 (2.5 cm × 40 ~m), followed by ion exchange chromatography on CM-cellulose with a gradient of increasing salt concentration from 0.005 M to 0.5 M ammonium acetate, pH 5.8 to 6.8. The fractions of the main protein peak were lyophilized and desalted by passage through a column of Sephadex G-25 equilibrated with 1 ~ acetic acid. The protein fractions were then pooled and lyophilized.

Identification of arginine residues modified by phenylglyoxal In order to differentiate the functionally important arginine residues in the amino acid sequence of cobrotoxin, the toxin was reacted with phenylglyoxal at varying pH and the modified derivatives were reduced and S-carboxymethylated by the procedure described by Crestfield et al. [14], followed by chymotryptic digestion. The S-carboxymethylated derivatives were dissolved in 0.1 M NH~HCOs buffer (pH 8.2) to give a 1% solution, and chymotrypsin was added (50:1). Digestion was carried out at 27 °C for 5 h and the digest was dried over PzO5 in a desiccator under vacuum. Arginine-containing peptides from chymotryptic digests were separated by a combination of high voltage paper electrophoresis at pH 5.4 with pyridine-acetic acid-water (20:7:973, by vol.) and descending paper chromatography with n-butanolacetic acid-water-pyridine (15:3 : 12:10, by vol.), as previously described [6]. Peptides on the map were developed initially with 0.2 % ninhydrin in acetone and thereafter with Sakaguchi reagent to detect arginine-containing peptides. In this procedure, the arginine-containing peptides from chymotryptic digests could be completely separated from each other and obtained in a good yield.

Amino acid analysis About 0.2/~mole of protein sample was hydrolyzed in 1 ml of constant-boiling HCI (5.7 M) at 1I0 °C for 24 h in evacuated sealed tubes. Amino acids were determined on a Technicon amino acid autoanalyzer, using norleucine as an internal standard.

Measurements of lethal activity Lethality was measured by intraperitoneal injection of a serial 2-fold diluted toxin solution into mice (16-18 g) with the amount of 0.2 ml per mouse, as previously described [15]. Six mice of both sexes were used for each dilution, and the LDso was calculated according to the 50 % end-point method of Reed and Muench [16].

Immunological procedures Double diffusion in agar gel was performed by Ouchterlony's technique [17] as previously described [2]. The quantitative precipitation reactions were carried out as described by Kabat and Mayer [18]. Increasing amounts of antigen in 0.02 M Tris-HC1 buffer (pH 7.5) containing 0.15 M NaCI were added to a constant amount

4 of antisera in a total volume of 1 ml. The tubes were incubated for 30 min at 37 C and then left overnight at 4 °C. The precipitates were washed 3 times with cold 0.15 M NaCI, after which they were dissolved in 3 ml of 0.1 M NaOH, and the absorbances were measured at 280 nm. RESULTS

Chemical modification of arginine residues in cobrotoxhl Changes of lethal activity of cobrotoxin by reaction with phenylglyoxal at varying pH values are shown in Fig. 2. The lethal activity decreased rapidly when the

[00~

pH6.0 ,.

40

"~.

20

40

60

80

TIME (min) ~i8. 2. Chanses o£ lethal activity of cobrotoxin by reaction with phenylslyoxal a~ varyin6 pH values. 6 m8 of cobrotoxin was dissolwd in 0.5 ml of buyer solution and a I ~ - f o l d molar excess of phenyl81yoxa] in 1.5 ml of the same buyer was added. Reaction was allowed to proc~d at 27 °C. After suitable intervals of time, aliquots were taken for determination of lethal activity. BuEer solutions used were: 0.1 M acetate, pH 6.0; 0.1 ~ phosphate, pH 6.7 or pH 7.5 and 0.2 ~ N-ethylmorpholine acetate, pH 8.0.

reactions were performed at pH 7.5 or higher pH and the lethality lost almost completely after 60 rain. However, the rate of inactivation was decreased significantly when the pH of the reaction was lowered. The lethal activity remained unchanged when the reaction was proceeded at pH 6.0 even for 80 min.

Identification of arginine residues modified by phenylglyoxal The results of amino acid analysis of the modified derivatives (Table I) showed that four of the six Arg-residues in cobrotoxin were modified when the reaction was carried out at pH 8.0. However, only one Arg-residue was modified when the reaction was carried out at pH 6.0 and essentially two and three Arg-residues were modified by the reactions at pH 6.7 and pH 7.5, respectively. All other amino acids remained essentially unchanged. In order to determine the position of the modified Arg-residues, the modified derivatives were digested with chymotrypsin after reduction and S-carboxymethylation. Peptide maps prepared from chymotryptic digests by a combination of high voltage paper electrophoresis and paper chromatography were compared with that of native toxin (Fig. 3). The Arg-containing peptides were detected by Sakaguchi reagent and the position of Arg-residue which was selectively modified with phenyl-

TABLE I AMINO ACID COMPOSITION OF COBROTOXIN AND ARGININE-MODIFIED DERIVATIVES Amino acid

Residues per mole of protein _

_

Cobrotoxin Modified derivatives

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan

8 8 4 7 2 7 . 8 1 . 2 1 2

. .

3 2 6 1

pH6.0

pH6.7

pH7.5

pH8.0

8.1 7.8 3.9 7.0 1.8 7.1 . 8.1 0.9 . 1.9 1.0" 2.0 -3.0 2.0 5.1 1.0

8.0 7.9 3.8 7.1 1.8 7.2 . 8.0 1.0 . 2.0 1.0 ! .9 -2.9 1.9 4.1 1.0

8.1 7.9 3.9 7.1 1.9 7.0

8.0 7.9 4.0 7.0 1.7 7.1

7.9 0.9

7.9 1.0

2.0 1.0 2.0

1.9 1.0 1.9 -2.9 1.9 2.1 1.0

3.0 1.9 2.9 1.0

* All values are expressed as molar ratios based on leucine = 1.0. glyoxal at p H 6.0 was identified as Arg-28 (Fig. 3-B). Two o f the six Arg-residues which were modified at p H 6.7 were identified as Arg-28 and Arg-33 (Fig. 3-C). It can be seen in Fig. 3-D, an additional Arg-residue modified at p H 7.5 was Arg-30. As shown in the peptide m a p (Fig. 3-E) o f S-carboxymethylated-Arg-modified toxin at p H 8.0, three Arg-59 containing peptides (C-1, C-3 and C-11, see Table II) and one Arg-36 and Arg-39 containing peptide (C-13, see Table II) are positive for Sakaguchi test. The result o f amino acid analysis (Table I) shows that four Argresidues were modified. This suggests that either Arg-36 or Arg-39 was also modified besides Arg-28, 30 and 33. Therefore, the Arg-36 and Arg-39 containing peptide, C-13, was cut out, eluted with 1 M acetic acid and dried. The dried material was dissolved in 0.2 ml o f 0.1 M N H 4 H C O a (pH 8.2) and 0.05 ml o f trypsin solution (5 mg/ ml) was added. After the mixture was incubated at 27 °C for 2 h, the p H o f the solution was lowered. The solution was dried for paper electrophoresis at p H 5.4 along with authentic arginine, asparagine and glutamic acid. The paper electrophoretogram shown in Fig. 4 gave three spots; two at cathodic side, o f which one spot corresponds to asparagine, and one at anodic side. N o free Arg-residue was present. Since the peptide C-13 has the following amino acid sequence (see Table II), 36 39 48 Arg-Thr-Glu-Arg-Gly-Cys-Gly-Cys-Pro-Ser-Val-Lys-Asn

¢

T

T

RCM-Cobrotoxin c-5

C-14 .

~ -

c-8 O

C-12

0

c - ~~,A r g -c-~5 o~ 30,33

O

C-7

Arg-~rg_28,30

C-2

c_~O ~-~b0

C-11~ Arg-59

C- 00

C-13~ Ar9-36,39

C-6 C-3 C-~

() 0~o~0 (}

~

+

pH 5.4, $0 V/cm, 90 min (1 st) Pyridine-Acetic acid-Water (20 : 7 '- 973)

•

(A~

pH 6.0

Arg-33

O

O

Arg-28,30

0

@@~ro_~o,.

O

0

0

0 Ar~_~ 0

Arg-59 e

[)o ~,~

hrg-36,39~

+ (B)

pH 6 . 7

©

O

0

Arg-28,30

O

0

~Ar~-.,.

ArN-59~ Ar~q-36,390

O

O

Ar~-59O

3 o ~',~.~ +

J

(c)

pH 7,5

O o

0

O O

O

0 Arg-59~ Arg-SPO

r•6

A g-3 ,39

DO

~rg-~9

--

+

(D)

pH 8.0

0

0 0

0

0

0

Arg9, Q hr~-36,39 -

rg-

O2, Arg-59 +

(E) Fig. 3. Peptide maps of the chymotryptic hydrolysates of reduced and S-carboxymethylated (RCM) cobrotoxin and Reduced and S-carboxymethylated modified derivatives at varying pH. S-carboxymethylated cobrotoxin (A); reduced and S-carboxymethylated Arg-modified toxins: at pH 6.0 (B); at pH 6.7 (C); at pH 7.5 (D); at pH 8.0 (E).

and if only Arg-36 was modified, the following two peptides and a free asparagine will be given off after tryptic digestion, 36 39 Arg-Thr-Glu-Arg Gly-Cys-Gly-Cys-Pro-Ser-Val-Lys Asn of which Arg-39 containing peptide gives a positive color reaction with Sakaguchi

TABLE 11 AMINO ACID SEQUENCE OF CHYMOTRYPT1C PEPTIDES FROM REDUCED AND S-CARBOXYMETHYLATED COBROTOXIN Peptides

Amino acid sequence

C-I C-2 C-3

H-Cys-Cys-Thr-Thr-Asp-Arg-Cys-Asn-Asn-OH Arg-59 H-Gly-Cys-Ser Gly-Gly-Glu-Thr--Asn-Cys-Tyr-OH H-Gly lle-Glu-lle-Asn-Cys-Cys-Thr-Thr-Asp Ar~g-Cys Asn-OH Arg-59 H-Thr-Gly-Cys-Ser-Gly Gly Glu-Thr-AsnCys-Tyr-OH H-Cys-Tyr-O H H-Asp-OH H-Leu-Glu-Cys-His-OH H-Gly-lle-Glu-lle Asn -OH H-Leu-Glu-Cys-His-Asn-GIn-GIn-OH H-Arg-Cys-Asn-Asn-OH Arg-59 H-Ser-Ser-Gln Thr-Pro-Thr-OH H-Arg Thr-Glu-Ar~g-Gly Cys-Gly-Cys-ProSer-Val-Lys-Asn-OH Arg-36 and 39 H-Arg-Gly-Tyr-OH Arg-33 H Arg-Asp His-Arg-Gly-Tyr-OH Arg-30 and 33 H-Lys-Lys--Arg-Trp-~rg-Asp-OH Arg-28 and 30 H-Lys-Arg-Trp-OH Arg-28 H-Lys- Lys--Arg-Trp-OH Arg-28

C-4 C-5 C-6 C-7 C-8 C-9 C- I I C- 12 C-13 C- 14 C-15 C-16 C- 17 C- 18

_

Arg-residues

_

e

o

0

Tryptic digests of C-13

• Arg

o

AS~

O

Glu

~

--

Start .

÷

pH 5.4, 4.0 V/cm, 7 0 rain • Pyridine-Acefi¢ acid-Wa~er (~O : 7 : g73~

Fi~. 4. Paper ele~rophore~o~r~mof the trypti¢ d i ~

of C-I~ (Ar~-~6 and ~9 ¢onminin~ pepfide)

from chymotryptic hydrolysates of reduced and S-carboxymethylated Arg-modified cobrotoxin (at pH 8.0). reagent. Indeed, it h a p p e n e d and the results indicate a c c o r d i n g l y t h a t Arg-36 was also modified besides Arg-28, 30 a n d 33 at p H 8.0. '

Characterization of the modified derivatives As shown in Fig. 5, the ultraviolet a b s o r p t i o n spectrum o f c o b r o t o x i n c h a n g e d extensively by reaction with phenylglyoxal. However, only Arg-28 modified derivative showed a similar s p e c t r u m a r o u n d 280 nm as t h a t o f native toxin.

i.0

~ z

0.8

a. o

0.6

Arg-~8.30,33 a ;56

/

N ,~ 0.2 O

cb ~ 240

~ 260

I 280

I ~00

W~VELENGTH

~ t 320

540

(nm)

Fig. 5. Absorption sp~tra of cobrotoxin (Cbt) and the Arg-modified derivatives. Each sample (2 mg) was dissolved in 4 ml of 0.01 M ammonium bicarbonate. Arg-28, represents the Arg-28 modified toxin; Arg-28 and 33, Arg-28 and 33 modified toxin; Arg-28, 30 and 33, Arg-28, 30 and 33 modified toxin; Arg-28, 30, 33 and 36, Arg-28, 30, 33 and 36 modified toxin.

Almost complete loss of lethal activity (Table III) and pronounced decrease of antigenic activity (Figs 6 and 7) were observed when four Arg-residues, Arg-28, 30, 33 and 36, in cobrotoxin were modified. However, Arg-28 modified derivative retained essentially full lethality and antigenic activity, suggests that the Arg-28 which is most accessible to phenylglyoxal modification is not essential for biological functions of cobrotoxin. The lethal activity of Arg-28 and 33 modified toxin drops precipitously, but the antigenic activity was not altered significantly. However, the lethality lost almost completely and the antigenic activity decreased 30 ~o when an additional Arg-residue at position 30 was modified. These results indicate that Arg-30 and Arg-33 are functionally important for the lethal activity and Arg-30 and Arg-36 are more closely related to the antigenic specificity of the toxin. In order to determine whether the Arg-modified derivative forms a polymer, gel filtration on Sephadex G-50 was performed (Fig. 8). In comparison with cobrotoxin (mol. wt, 6949) and a-chymotrypsin (mol. wt, 22 000), both the Arg-28 and 33 modified toxin and Arg 28, 30, 33 and 36 modified toxin revealed significant amount of polymerization and the amount of polymerized forms increased in parallel with the degree of Arg-modification. After gel filtration of Arg-28 and 33 modified toxin, the fractions emerged in the same elution volumes as those of cobrotoxin and aTABLE III STEPWISE MODIFICATION OF A R G I N I N E RESIDUES IN COBROTOXIN WITH PHEN Y L G L Y O X A L AT VARYING pH

Cobrotoxin pH 6.0 pH 6.7 pH 7.5 pH 8.0

Arg-residues modified

Lethality (~)

Antigenic activity ( ~ )

None Arg-28 Arg-28 and 33 Arg-28, 30 and 33 Arg-28, 30, 33 and 36

100 100 22.6 3.1 1.6

100 98 94 70 34

lO Q25 pH 6.0

Cbf

~.:'-'~'1~": ~"~ H °

~ ~ o.~ o

Z// l/~

~

~~ o.lo

,pHi5 ~'x

~'~

~72,

~ ~5

m

I I 0

~ 20

I 30

I 40

5~0

ANTIGEN (~g)

Fig. 6. Quantitative ~recipitation reactions of cobrotoxin (Cbt) and the Arg-modi~ed derivatives with anti-cobrotoxin serum. 0.4 ml of antiserum was used in each case. ~ , cobrotoxin; ~ ~ Arg-28 modified toxin (at pH 6.0); z ~ . , , Arg-28 and 33 modified toxin (at pH 6.7): ~ , ~ x , Arg28, 30 and 33 modified toxin (at pH 7.5): ( , - ~), Arg-28, 30, 33 and 36 modified toxin (at pH 8.0). ~

~

Fig. 7. lmmunodiffusion in agar gel. Central well: anti-cobrotoxin serum. Surrounding wells: C, cobrotoxin; 6.0, Argo28 modified toxin (at pH 6.0); 6.7, Arg-28 and 33 modified toxin (at pH 6.7); 7.5, Arg-28, 30 and 33 modified toxin (at pH 7.5); 8.0, Arg-28, 30, 33 and 36 modified toxin (at pH 8.0). c h y m o t r y p s i n , c o r r e s p o n d i n g to the m o n o m e r i c a n d p o l y m e r i z e d forms were pooled for the d e t e r m i n a t i o n o f biological activities. The lethality and antigenic activity were measured after the fractions were desalted a n d lyophilized. A s shown in Table IV, the lethal activity a n d antigenic activity o f the m o n o m e r i c form is a l m o s t the same as those o f the modified derivative. A l t h o u g h no p r o n o u n c e d decrease o f antigenic activity is noted, the p o l y m e r i z e d form is c o m p l e t e l y non-toxic.

11

~

E I0

:, ;~ * ~,,~

~

o ~ '~

tt

B

~

i

~

~

~ Z

I~ 4

0

I

-

I0

~

~0

20

FRACTION

~

40

I

50

NUMBER

Fig. 8. Gel filtration patterns of cobrotoxin, Arg-modified derivatives and a-chymotrypsin on Sephadex G-50. The column was equilibrated with 0.067 M phosphate buffer (pH 7.4) to a constant height (2 cm × 83 cm). Each sample (10 mg) dissolved in the same buffer, was applied onto the column and eluted with the same buffer. 5 ml fractions were collected at a rate of 25 ml per h and the protein concentration was determined by the method of Lowry et al. [19]. ~ - - ~ , cobrotoxin; × - - ×, Arg-28 and 33 modified toxin; A--A, Arg-28, 30, 33 and 36, modified toxin; 0---0, tt-chymotrypsin. TABLE IV The lethality and antigenic activity of monomeric and polymeric forms separated from Arg-28 and 33 modified toxin by gel filtration on Sephadex G-50.

Cobrotoxin Arg-28 and 33 modified toxin Monomeric form Polymerized form

Lethality ( ~ )

Antigenic activity (%)

100

100

22.6 25 0

94 95 78

DISCUSSION Arg-28 was f o u n d to be the m o s t accessible to the reaction with phenylglyoxal, a n d it is the only one modified at p H 6.0 w i t h o u t a l t e r a t i o n in biological activity o f c o b r o t o x i n . Arg-33 is the next one modified when the reaction was carried o u t at p H 6.7. A n a d d i t i o n a l residue modified at p H 7.5 was f o u n d to be the Arg-residue at the p o s i t i o n 30. Arg-30 a n d Arg-33 are i m p o r t a n t for the lethal activity a n d Arg-30 a n d Arg-36 are m o r e closely related to the antigenic specificity o f the toxin. There are three moles o f arginine in toxin a a n d t w o in toxin b o f Laticauda semifasciata. O n l y one A r g - r e s i d u e was modified in b o t h toxins after reaction with 1,2-cyclohexanedione [20]. T h e modification d i d n o t alter the lethality o f the toxins, confirms t h a t at least one o f the Arg-residues is n o t essential for the lethal activity o f snake neurotoxins. Siamensis 3 toxin o f Naja naja siamensis [21] contains five arginine residues at the p o s i t i o n s 2, 33, 37, 68 a n d 70. Digestion o f the toxin with p r o t e a s e f r o m Arthrobacter r e m o v e d the C - t e r m i n a l t e t r a p e p t i d e , A r g - L y s - A r g - P r o , a n d the resulting molecule is h a l f as toxic as the intact toxin. The result indicates t h a t neither

12 Arg-68 nor Arg-70 is essential for lethality. However, the toxin was inactivated by reaction with phenylglyoxal and the inactive monomeric derivative, having only one arginine residue modified, was separated. Pharmacological properties of postsynaptic neurotoxins are the same as those of D-tubocurarine, except for the slowness of onset and the lower reversibility of the paralysis [22, 23]. The latter difference is probably due to the larger molecular size of basic polypeptides in comparison to D-tubocurarine. Both short and long neurotoxins [24] show a great affinity to the acetylcholine receptor on the motor endplate [25-29]. Although one cannot at present, exclude the possibility that the apparently irreversible toxin-receptor interaction might involve the formation of a covalent bond, for example, by disulfide interchange, it still seems likely that the cationic groups present in snake neurotoxins might be directly involved in the interaction with the receptors. Probably, as in the case of o-tubocurarine, at least two basic groups with positive charge held at a certain distance in the molecule may be responsible for their neuromuscular blocking activity. Chemical modification of Lys-47 with group specific reagents resulted in complete loss of lethal activity without change of the conformational properties [9, 30], this suggests that the positive charge contributed by the e-amino group of Lys-47 (or guanidino group of arginine residue) which is present in all sequence of snake neurotoxins (Fig. 9) is functionally important for the biological activity of neurotoxins. The results of selective and stepwise arginine modification with group specific reagent, phenylglyoxal, at varying pH suggest that at least the Arg-residue at position 28 in cobrotoxin is not essential. However, when an additional Arg-residue at position 33 was modified the lethal activity dropped precipitously, and the lethality was even lost almost completely when a third Arg-residue at the same region was modified. This indicates that the cationic groups contributed by guanidino groups of Arg-residue are also functionally important. This functionally important cationic group located in the loop containing the sole Trp-residue: namely, the region between positions 25 and 40 (Fig. 1) which contains most of the basic residues and aromatic functional residues, and this uncross-linked loop possibly protrude outward from the molecule because of its hydrophilic properties. This result, together with previously published works [9, 30] on the chemical modification or removal of cationic groups, tend to indicate that no individual cationic group is essential. The reason for the appreciable (22.6~o) residual activity of the Arg-28 and 33 modified derivative might be due to reversal of the derivatization in vivo or prior to assay, since the reaction is slowly reversible at neutral pH [13]. All postsynaptic neurotoxins sequenced so far have a sequence Arg-Gly occurring at the same or homologous positions (Fig. 9). lfthere should exists an additional functionally important cationic group, this may be a guanidino group of Arg-33. Therefore, it seems likely that the positive charges of the e-amino group of Lys-47 and of the guanidino group of Arg-33 probably form salt bridges with the anionic sites of the receptors which recognize the quaternary amonium ion of acetylcholine. The definite answer will have to await the isolation of the toxin receptors and the identification of the functionally important basic residues which are masked to produce selective chemical modification in the toxin-receptor complex, and the structure analysis by X-ray crystallography may provide a basis for consideration of structure-function relationship for snake neurotoxins and the studies are now under progress.

melanoleuca nivea halmah hannah jamesonii polylepis polylepis multicinctus

(toxin 3) (black cobra toxin) (toxin 3) (toxin ~) (toxin A) (toxin B) (toxin C) (toxin b) (toxin ~) (toxin a) (toxin b) (toxin II) (toxin ~) (toxin ~) (~-bun~arotoxin)

Gln-~rp~Asp-His,

%rg

,Gly~Lys .Gly{Lys .Gly{Lys .Gly{Lys .Gly~Lys .Gly{Lys .Gly{Lys Gly~Lys Gly{l,ys Gly{Lys Gly{Lys Gly{Lys ~rg Gly~Lys ~rg Gly{Lys krg Gly{Lys

Arg Val-Asp-Leu-Gly-CySArg Val-Asp-Leu-Gly-CySArg Val-Asp-Leu-Gly-CySArg Val-Asp-Leu-Gly-CySArg Val-Asp-Leu-Gly-CySArgVal-Asp-Leu-Gly-CySArg Val-Asp-Leu-Gly-CySArg Val-Asp-Leu-Gly-CySAr@ Val-Asp-Leu-Gly-CySArgVal-Asp-Leu-Gly-CySArg. lle-Asp-Leu-Gly-CySArg. Val-Glu-Leu-Gly-CySArg. Val-Glu-Leu-Gly-CySlle-Val-Glu-Leu-Gly-CySVal-Val-Glu-Leu-Gly-CyS-

-

Gly-

-CyS-Pro~'~Val

Thr-GlyThr-GlyThr-GlyThr-GlyThr-GlyThr-GlyThr-G1yPro-GlyPro-GlyPro-31yPro-GlyThr-GlyAla-GlyAla-GlyPro-Tyr-

Lys, Ser-GlyLys, Pro-GlyLys, Pro-GIyLys, Pro-GlyLysPro-GlyLysSer-GlyLyg Ly~ Arg Arg Arg ~rg Ly~ Lys Lys Lys Lys -CyS-Pro[Lys~VaI Lys -CyS-Pro~Lys~Val Lys -CyS-Pro~ Lys -CyS-Pr0-Ser~Lys Lys

-CyS-Pro-Thr-Val -CyS-Pro-Thr-Val -CyS-Pro-Tb-r-Val -CyS-Pro-Thr-Val -CyS-Pro-Thr-Val -CyS-Pro-Thr-Val -CyS-Pro-Thr-Val -CyS-Pro-Thr-Val -CyS-Pro~Val -CyS-Pro-Ile-Val

ArEIGly-Thr-Ile-Thr-Glu, Arg,.Gly-CyS-Gly-CyS-Pro-Gln-Val, Arg Arg ~rg Arg Arg Arg Arg ~rg ~rg ~rg ~rg

~

GlyArg Lys~GlyArE Arg, Lys~Lys~GlyArg, Lya Pro-GlyLys, Pro-GlyArE Lys. L y ~ G l y Arg. Arg,Gly-CyS-Gly-CyS-Pro~Val.Lys. Pro-GlyArg. Lys, Gln-Gly.Gly-Thr~Ile-Glu Arg, Lys. Pro-GlyArg, Lys. Ser-GlyArg, Ly~.Ly~Gly-

GIy-CyS-GIy-CyS-Pro-Ser-Val Ar~ Gly-CyS-Gly-CyS-Pro-Ser-Val, Arg Gly-CyS-Gly-CyS-Pro-Thr-Val, Arg Gly-CyS-Gly-CyS-Pro-Ser-Val, Arg Gly-CyS-Gly-CyS-Pro-Thr-Val. Arg Gly-CyS-Gly-CyS-Pro-Thr-Val, Arg Gly-CyS-Gly-CyS-Pro-Ser-Val, At6 Arg GIy-CyS-GIy-CyS-ProL~Val, Arg ~ly-CyS-Gly-CyS-Pro-Gln-Val, Arg ,Gly-Thr~Arg~lle-Glu GIy-CyS-GIy-CyS-Pro-GIn-Val, Arg ,Gly-Thr~Arg#Ile-Glu GIy-CyS-GIy-CyS-Pro-GIn-Val, Arg ,Gly-Thr~Arg~Ile-Glu, Ar E • GIy-CyS-GIy-CyS-Pro-GIn-Val, Arg~Gly-Thr-lle-lle-Glu. Arg, iGly-CyS-Gly-CyS-Pro-Thr-Val, ~rE#Gly-Thr-Ile-Ile-Glu, Arg,~Gly-CyS-Gly-CyS-Pro-Thr-Val.

6 .Gly-Tyr~Thr-Glu -Gly-Ser-Ile-Thr-Glu .Gly-Thr-Ile-Ile-Glu .Gly-Thr-Ile-Ile-Glu .Gly-Thr-Ile-Ile-Glu .Gly-Ser~'~Thr-Glu ,Gly-Thr-Ile-Ile-Glu ,Gly-Thr-Ile-Ile-Glu ,Gly-Thr-Ile-I]e-Glu

Gln-Trp-Ser-Asp-Phe, Gln-Trp-Ser-Asp-Phe, ArE#Gly-Thr-Ile-Ile-GluArg,.Gly-CyS-Gly-CyS-Pro-Thr-Val. Gln-Trp~Asp-His, Arg~Gly-Thr-Ile-Thr-GluArg,.Gly-CyS-Gly-CyS-Pro-Thr-Val,

Gln-Trp-Ser-Asp-Phe,

Ar--~Trp~Asp-His Tyr-TrpA~Asp-His, Thr-Trp-Ser-Asp-His Thr-Trp-Ser-Asp-His, Thr-Trp-Ser-Asp-His Thr-Trp-Ser-Asp-His Thr-Trp-Ser-Asp-His.

-Tyr-Thr Lys Thr-Trp-CyS-Asp- -Tyr-Thr Lys Thr-Trp-CyS-Asp- -Tyr-Thr Lys Thr-Trp-CyS-Asp- -Tyr-Thr Lys Thr-Trp=CyS-Asp- -Tyr-Thr Lys Thr=Trp-CyS=Asp- -Tyr-Thr Lys Thr-Trp-CyS-Asp- -Tyr-Thr Lys Thr-Trp-CyS-Asp- -Tyr-Thr Lys Thr=Trp-CyS-Asp- -Tyr-Thr Lys Met-Trp-CyS-Asp- -Tyr-Thr-Glu-Thr-Trp-CyS-Asp- -Tyr-Thr~Thr-Trp-CyS-Asp-Tyr-Thr#Lys#Thr-Trp-CyS-Asp-Tyr-Thr|Lys~Thr-Trp-CyS-Asp-Tyr-Thr~Lys#Thr-Trp-CyS-Asp-Tyr~Arg~Lys~Met-Trp-CyS-Asp-

-Tyr .Lys -Tyr ,Lys -Tyr ,Lys -Tyr ,Lys -Tyr .Lys -Tyr .Lys -Tyr Lys -Tyr-A~n,L7s -Tyr-His Lys -Tyr-His Lys -Tyr-Asn Lys -Tyr-Asn Lys

28 30 -Tyr~ ,~Trp~'~Aap-Hi s -TyrILya~Lya.~ T r p [ A r g # A s p - H i s -Tyr[LystLys.. V a l - T r p A r ~ A s p - H i s -Tyr~Lys~Lys,,Gln-Trp-Ser-Asp-His -Tyr-Asnt.Lys., A r g ~ T r p ~ = ~ A s p - H i s -Tyr~.Ly~ .Gln-Trp-Ser-Asp-His

Fig. 9. Amino acid sequence of snake neurotoxins showing the region between positions 25 and 49 which contains most of the basic residues.

N. N. 0. 0. D. D. D. B.

N. naja N. naja

siamensis naja naja naja

naja naja naja naja naja

N. N. N. N. N.

L. L. L.

L.

naja atra (cobrotoxin) haje haje ~, N. nivea ~ nigricollis (toxin ~) melanoleuca (toxin d) haemachatus (toxin II) haemachatus (toxin IV) nivea (toxin ~) polylepis (toxin ~) jamesonii (V~I) schistosa (toxin ~) schistosa (toxin 5) cyanocinctus (hydrophitoxin a) cyanocinctus (hydrophitoxin b) semifasciata (erabutoxin a) semifasciata (erabutoxin b) semifasciata (erabutoxin c) laticaudata (laticotoxin a) colubrina (laticotoxin a)

N. N. N. N. H. H. N. D. D. E. E. H. H. L.

14 ACKNOWLEDGEMENTS T h i s w o r k was s u p p o r t e d by the U.S. A r m y R e s e a r c h a n d D e v e l o p m e n t G r o u p ( F a r East), D e p a r t m e n t o f the A r m y u n d e r G r a n t N o . D A - R D R F - S 9 2 - 5 4 4 - 7 3 - G 194, D A - R D R F - S 9 2 - 5 4 4 - 7 4 - G 2 1 0 , a n d by t h e N a t i o n a l Science C o u n c i l , T a i w a n . REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

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