Toxtcow, 1976, Vo4 14, pp. 295-306. ParQamon Prey . Printed in C3reat Britain.

NITRATION OF TYROSINE IN THREE COBRA NEUROTOXINS EVERT 11AAL 7AlJN and JOHAN SUNDELIN Institute of Biochemistry, Box 576, S-751 23 Uppsala, Sweden (Acceptedjor publication 15 December 1975) E. Krutlssox and J. Stlrrn>?l.lx. Nitration of tyrosine in three cobra neurotoxins . Toxkon 14, 295-306, 1976.-The curarumimetic snake venom toxins always have a tyrosine residue in the same position in their sequences. This invariant tyrosine in three neurotoxins has been nitrated with tetranitromethane. The toxins were : (1) siamensis 3 of the structural type 71-5 (no. of amino acid residues and disulfides) and with one tyrosine, the main ncurotoxin of the venom of Naja naja sfamensis, (2) siamensis 7C (62~ and two tyrosines), a minor neurotoxic component of the same venom, and (3) toxin a (61-4 and one tyrosine), the main neurotoxin of Naja nigrkollis . The first toxin is a so-called long neurotoxin (a toxin with five disulfides and ca. 70 amino acid residues) and the other two short toxins (four and ca. 60). The 3-nitrotyrosyl derivatives of siamensis 3 and toxin a were isolated by gel filtration on Sephadex G-50 and ion-exchange chromatography on Bio-Rex 70 . Both derivatives had i.v . l.nlaa -doses of 150 ltg per kg mouse, corresponding to 67 ~ of the initial toxicity. This high residual activity indicatesthat the invariant tyrosine does not have an essential role neither as a stabilizer of the active conformation nor as a functional group. The nitration of toxin 7C involved the modification of both tyrosine residues and was accompanied by great structural chaagea and loss of activity. The inactivation does not imply that the invariant tyrosine should bo functionally essential in this toxin, but it is rather a consequence of the serious conformational changes accompanying the modification . The p%pp of the invariant tyrosine is 10'5 in siamensis 3 and 116 in toxin a and probably about the same in toxin 7C . This indicates that this tyrosine in the long neurotoxin has an exposed position, whereas it is in the hydrophobic interior of the two short toxins, or that its ionisation is greatly affected by neighbouring groups. In the discussion it is pointed out that the very strong binding between a neurotoxin and its target, the acetylcholine receptor is probably a result of the interaction between several groups in the toxin and the receptor. The majority of amino acid residues with reactive groups in the side chains have been modised with retention of a significant fraction of the initial activity. It seems likely that at least one of these groups (amino, carboxyl, tyrosinyl, tryptophanyl) should be in contact with the receptor in the toxin-receptor complex. If so, that should imply that it is possible to chemically modify such an interacting group; the interaction from the remaining groups should still be sufiïcient to bind the toxin to its target . There exists a great variety of cholinergica, which have only one obvious feature in common, a strongly positively charged group, which is suggested to be a recognition site, the function of which is to make it possible for the molecule to recognize the target . An invariant arginine residue is assumed to provide the recognition site in the neurotoxins. INTRODUCTION

ctrxnltlNn~c snake venom toxins have an invariant tyrosine residue as a part of the sequence Cps-Tyr. The conservation of tyrosine in this position should be important in some way, otherwise it is difficult to understand why no mutations have occurred there, but have been frequent in most other places in the molecule (Fig. ~. The effects of modification of the invariant tyrosine have been studied in several cases. The cobrotoxin of Naja naja else, structural type 62-4 (no. of amino acid residues and disulfides) which has two residues of tyrosine, was treated with tetranitromethane . The

TxE

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EVERT I{ARLSSON and JOHAN SUNDELIN

extra tyrosine was readily nitrated without loss of toxicity. The invariant tyrosine could not be nitrated until the molecule was unfolded with 5 M guanidine hydrochloride, and the extra tyrosine was thereby also modified . The completely nitrated toxin was inactive. It had a greatly different circular dichroism spectrum, and it did not react any longer with the antiserum against the native toxin (CHANG et al., 1971a). This indicates that the complete nitration caused great structural changes, and the loss of toxicity appears therefore to depend on these changes rather than on the modification of a functionally essential site. Iodination in the absence of guanidine hydrochloride of both tyrosines in cobrotoxin was done with preservation of the activity and almost full reactivity with anticobrotoxin sera (HunxG et al., 1973). This contradictory result suggests that iodination is a milder method not associated with any structural changes with serious effects on the activity. Nitration of the invariant tyrosine of neurotoxin T of Naja haje, structural type 61-4 with only one tyrosine, without any denaturing agents present resulted in the formation of 083 residues of 3-nitrotyrosine per molecule and a decrease in toxicity of 84~ (CHICHEPORTICHH et al., 1972). The decrease in toxicity parallel to the formation of nitrotyrosine might suggest that the nitration cannot be done without inactivation . Modification of a similar neurotoxin (structural type 61-4 and one tyrosine) from the sea-snake Lapemis hardx~ickü was also carried out without denaturation . Both iodination to 84 ~ and nitration to 50 ~ were accompanied by large decreases in toxicity (RAYMOND and Tu, 1972). The nitration of the invariant tyrosine in a long neurotoxin (structural type 71-5) from the Indian cobra Naja naja could be done with retention of 80 ~ of the original activity (OHT'A and IIAYASHI, 1974). This indicates that the invariant tyrosine is not functionally essential . These apparently contradictory results prompted us to reinvestigate the effects of the nitration of the invariant tyrosine. We also considered it worthwhile to isolate as far as possible a monomerc derivative of the nitrated toxins and study its properties, instead of investigating a sample which might contain both monomers and differs. This precaution was not taken in any of the previous cases. The following toxins were investigated : (1) siamensis 3 (structural type 71-5 and one tyrosine), the main neurotoxin of Naja naja siamensis (KARLSSON et al., 1971), (2) toxin a (structural type 61-4 and one tyrosine), the main neurotoxin of Naja nigricollis (KARISSON et al., 1966), and (3) siamensis 7C (62-4 and two tyrosines), a minor neurotoxic component of the siamensis venom (KARLSSON et al., 1971) . Toxin 7C is a close homologue of the cobrotoxin of Naja raja atra . It differs from cobrotoxin apparently only by an Arg -" Lys substitution . The first toxin is a so called long neurotoxin (toxins with five disulfides and about 70 amino acid residues) and the other two are short toxins (four and ca. 60). MATERIALS AND METHODS Neurotoxins The two neurotoxins of Ngja ngja siamensis were purified directly from the crude venom (Miami Serpentarium) by chromatography on Bio-Rex 70. Venom (2 g) was dissolved in 15 ml of 004 M ammonium acetate and applied to a 32 x 25 cm column of Bio-Rex 70 (equilibrated with 0'20 M ammonium acetate, pH 650), which had been pyre-eeluted with 20 ml of 008 M ammonium acetate. The column was then eluted with S column volumes of 008 M ammonium acetate followed by 0' 14 M solution until the main neurotoxin was eluted from the ion-exchanger. Under these conditions the main neurotoxin is directly obtained in a pure form . The column was eventually eluted with a concave gradient from 014 to 140 M ammonium acetate as previously described (I{nRrssox et al., 1971). The toxin 7C was then also obtained in a pure state. The toxin a of Ngja nigricollis was prepared by gel filtration of the crude venom on Sephadex G-7S and ion~acehange chromatography on Bio-Rex 70 equilibrated with 0'20 M ammonium acetate, pH 730. The freeze-dried neurotoxic fraction was dissolved in 005 M ammonium acetate and applied to the ion~xchange TOXICON1976 Vol. I~

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column pre-eluted with 0'OS M acetate . This was followed by elution with one bed volume of 0'OS M acetate and to desorb the toxin with a concave gradient from 0'09 to 1'40 M ammonium acetate. Nitration of neurotoxi'ns (RIORDAN et al., 1967)

Two to four Ilmoles of neurotoxin was dissolved in 2 ml of Tris buffer, pH 8'0 and S M in guanidine hydrochloride . The buffer was prepared by dissolving guanidine hydrochloride in 0'2 M Tris to the desired concentration and adjusting the pH . One hundred ul of 95 ~ alcohol containing tetranitromethane to give a 20-fold molar excess was added. The reaction was allowed to proceed at room temperature for 2 hr . Isolation of nitrated toxins

The reaction mixture was gel filtered on a column of Sephadex G-SO in 0'OS M ammonium acetate. The different protein fractions were screened for toxicity by injection of suitable aliquots into the tail vein of white mice . The toxic fraction was then applied to a column of Bio-Rex 70 equilibrated with 0'20 M ammonium acetate, pH 6'S0. A concave gradient was used to displace the toxin derivatives from the resin. The concave gradient used in the experiments was obtained by the Beckman gradient pump Model 131 programmed by a specialcam (Part No . 324812). The gradient can be approximates by a simple two cylinder device with an area ratio 1 '4 and with the output from the larger vessel . Toxlclty The LDloo-doses were determined by i.v . injection into white mice weighing between 1S and 20 g. Three mice were used at each dose level. The concentration of a sample was calculated from its absorbance and A~ I ~ coefficient, the absorbance of a solution having the concentration of 1 mg per ml, which was obtained by quantitative amino acid analysis of a suitable aliquot from a sample with known absorbance . Amino acid analysis

The samples were hydrolyzed with 6 M HCl at 110°C for 24 hr and the hydrolysates were analyzed on a Durrum D-500 amino acid analyzer .

Spectrophotometrlc titration

The sample was dialyzed either against 0'OS M ammonium acetate and 0'1 M NaCI (native toxins) or 0'OS M acetic acid and 0'1 M NaCI (nitrated toxins). One part was used as a reference solution, while the pH in the other part was varied by addition of minute amounts of S M NaOH. The pH and the change of absorbance at 294 nm (native toxins) or 430nin (nitrated toxins)were measured aftereach addition of alkali . The titrations were done at room temperature. RESULTS

Nitration of siamensis 3

Freeze-dried toxin (1T2 mg ; 2"2llmoles) was nitrated and then gel filtered on Sephadex G-50 (Fig. 1). The two first peaks contained protein (positive Folin reaction) and the material eluting after 240 ml consisted of low molecular weight reagents and buf%r constituents . A,K 1.0 0 .3

00

100

200

300

Effluent (ml)

FIG. I . GEL FILTRATION OF THE NrTRATED SIAMENSIS 3 ON SEPHADEX G-SO (2'O x 93'S cm, voro voLUM>; 9S ml) ne 0'OS M AMMONIUM ACETATE. Flow rate 18 ml per hr . Fraction size 3" 6 ml . The toxic material eluting between 145 and 185 ml

was recovered and chromatographed further on Bio-Rex 70 (Fig . 2) .

In a control experiment, the toxin was exposed for 2 hr to the Tris-guanidine hydrochloride buffer without tetranitromethane . The subsequent gel filtration on G-50 gave two peaks in the same position as in Fig. 1 . The first peak contained less than S ~ of the protein TOXICON 1976 YoJ . I~

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EVERT ICARLSSON and JORAN SLJNDELIN

and consisted of dimers normally present in a lyophilized preparation of the toxin and the second one was the monomerc form of the toxin (KA1tLSSOIV et al., 1971) . The two protein peaks in Fig. 1 should then correspond to dimers and monomers, respectively, ofthe nitrated toxin. It is also evident that a considerable dimerization had occurred as the first peak contained 25 ~ of the protein (actually 25 ~ of the absorbance). A~0

ro#

0.3

0?0 M

(~ EE 11 . The nitration lowered the pK considerably . The modified siamensis 3 had a pKe ~ of TOXICON 1976 Yd. I~

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EVERT I{ARLSSON and JORAN SUNDELIN

5~8 (Fig. 4b) and toxin a 6~9. Toxin 7C was after nitration dialyzed against 005 M acetic acid and 0~ 1 M NaCI. The sample therefore contained probably all the different forms of the modified toxin which give the complex gel filtration pattern shown in Fig. 3. However, only one stage in the ionization with a pK,Pp of 6~7 could be detected. en,

0.8 0.6 0.4 pK~pp 10.5 0.2 0~ 6

10

12

P

H 1~'

FIG . 4 . SPEGTROPHOTOMETRIC TITRATION OF TYROSYL GROUPS IN NEUROTOXINS.

A. Native siamensis 3. B. Nitrated siamensis 3. The titration curve shows 10 ~ protonation at pH 7~3-7~5 (pH of the blood) assuming complete titration at pH 11 . C. Native siamensis 7C. DISCUSSION

The nitration of the invariant tyrosine both in a long neurotoxin (siamensis 3) and in a short one (toxin a) can be done with retention of as much as 2/3 of the original activity. The results with siamensis 3 agree with those obtained by Ox~rA and HnYnsI-n (1974) in their investigation of a homologous long neurotoxin from the Indian cobra Naja raja. Our results with toxin a are contradictory to those of others (CI-ncx»oRTlcl~ et al., 1972 ; 1ZAYMOND and TU, 1972) in their experiments with homologous neurotoxins of the same TOXICON 1976 I'ol .

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Tyrosine Nitration in Cobra Neurotoxins

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structural type 61-4 and with only one tyrosine. In these cases, the nitration was accompanied by a parallel decrease in toxicity hinting that the nitrated toxins should be inactive . This discrepancy may depend on the toxins investigated in the particular cases, but we are, however, inclined to ascribe this to differences in the design of the experiments. Toxin a was unfolded with guanidine hydrochloride rendering the tyrosine easily accessible and a 20-fold molar excess of tetranitromethane was sufficient for a complete nitration. The other short toxins were nitrated in the absence of denaturants and in spite of using a much higher excess (400-10,000-fold) of the reagent a complete nitration was not achieved . But a high concentration of tetranitromethane favours aggregation (TSUKAMOTO and OFU~to, 1974) and in the two experiments a large amount of non-toxic dimers and polymers were probably formed and that could well explain the high degree of inactivation observed . But as no investigation, such as gel filtration, of the amounts of aggregates was done in the two referred cases, no definite answer can be given. The complete nitration of toxin 7C caused inactivation . The main reason for this seems to be the great structural disturbances accompanying the nitration as is evident from the gel filtration (Fig. 3) . This is in agreement with the results obtained with cobrotoxin (Cxnxo et al., 1971a) . Two tyrosine residues were nitrated in both cases. The cooperative effect of the modification of two residues should have a comparatively greater effect on the structure and consequently also cause a greater decrease in toxicity. We do not think, however, that this is the only explanation. This is certainly to a great extent contributable to structural features characteristic for toxins of the type 62-4 . An indication of the existence of such typical properties is that neurotoxins of the type 62-4 crystallize rather readily (YANG, 1965 ; Tnamtn and Axai, 1966 ; Tu et al., 1971) in contrast to most other toxins . The invariant tyrosine is adjacent to a disulfide bond, but otherwise its environment appears to be rather different in short and long neurotoxins. The pKa~ for the invariant tyrosine in toxin a is 11 "6 and probably about the same in toxin 7C. This indicates that the invariant tyrosine is in the hydrophobic interior of the short neurotoxins, or that its ionization is greatly affected by neighbouring groups . A plCgpp of 10" 5 in siamensis 3 indicates an exposed position for the invariant tyrosine, since this value is close to the pK-values of 9"75 and 10" 15 found for exposed tyrosines in ribonuclease unfolded with 6 M GuHCI. Tyrosine residues adjacent to disulfides were ascribed the higher value. The vicinity of a disulfide bond should even in 6 M GuHCI suffice to raise the pKfrom 9" 75 to 10 "15 (Noznxi and TANFORD, 1967). The introduction of a vitro group into the tyrosine residue alters both the size and the charge considerably . The tyrosine residue in the native toxin may be involved in a hydrogen binding, but at least in the nitrated siamensis 3 this can hardly be the case. The pK,vv is as low as 5~8 and at pH 7" 7" 5, the acidity of the blood and the supposed pH of the neurotoxic reaction, only about 10 ~ of the molecules have a protonated phenolic group and thus capable of providing a hydrogen for binding. In the nitrated toxin a about 25 ~ of the molecules are protonated at the same pH. In spite of these apparently drastic changes both siamensis 3 and toxina retain as much as 2/3 of the activity after nitration. It seems therefore unlikely that tyrosine in these two toxins should have an essential role either as a stabilizer of the active conformation or as a functional group. The experiment with toxin 7C is not directly comparable with the other two experiments as it involved the nitration of two tyrosine residues. A selective nitration of the invariant tyrosine should have been preferable, but as pilot experiments to establish suitable conditions would have required more material than was available, this was not done. TOXICON 1976 Vol. l~

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The functionally essential groups in neurotoxins of all structural types are certainly the same, and it seems therefore likely that the invariant tyrosine is not functionally essential in any neurotoxin. Tyrosine is invariant and part of the same sequence Cys-Tyr also in cardiotoxins, lytic factors, etc., non-neurotoxic snake venom proteins homologous to the neurotoxins (SzltYnoM, 1973 ; Tv, 1973 ; RYn~ty et al., 1973 ; ICAxrssoN, 1973 ; YnNG, 1974). This also suggests that this tyrosine is required for other reasons than neurotoxicity, but what these reasons are, is presently not understood . A great deal of work involving chemical modifications of neurotoxins have been done in several laboratories. In connection with this work we feel it justified to summarize the majority of these experiments here and attempt to draw the conclusions from them. The modification of the invariant tryptophan (CHICFIEPORTICHE et al., 1972 ; Knxt ssoty et al., 1973 ; CxANC and YnxG, 1973 ; Ox~rA and HAYASHI, 1974) as well as of the invariant tyrosine does not inactivate the toxins unless the modification is accompanied by serious structural disturbances . Six out of seven carboxyl groups in cobrotoxin ca.n be esterified with retention of the activity and the reactivity for the anticobrotoxin serum. The remaining carboxyl, the variant glutamic acid-21, is esterified after unfolding the molecule with guanidine hydrochloride . The modification of this normally unaccessible carboxyl is accompanied by great structural changes and consequently also by loss of toxicity (CHANG et al., 1971b). Acetylation, or carbamylation of all six amino groups in siamensis 3 reduces the toxicity by 97 ~. The remaining activity is, however, significant and not due to contamination as discussed elsewhere (KnxrssoN et al., 1972). The fully acetylated or carbamylated siamensis 3 is still a potent toxin, as its activity expressed on molar basis is about the same as that of n-tubocurarine chloride. Furthermore, a selective acetylation of any one of the six amino groups in siamensis 3 gives a derivative with a residual activity of 67 % (KAxtssoN et al., 1972) and with the same binding properties to skeletal muscle as the native toxin (LIBELIUS, 1974). The statement that lysine-53 (numbering according to Fig. 5) should be functionally essential (CxAxG et al., 1971 ; CHICIiEPORTICHE et al., 1972) does not seem to be valid. Trinitrophenylation of lysine-53 together with lysine-27 in cobrotoxin abolished the activity (CHANG et al., 1971b) but caused also almost a complete loss of reactivity with the antiserum against the native toxin, this indicating great structural changes . Dansylation of lysine-27 and -53 in toxin I (the same as toxin a, Fig . 5) of Naja haje decreased the activity by 92 ~ but did not completely inactivate the toxin (CHICHEPORTiCHE et al., 1972) . Acetylation of lysine-53 in toxin a (In of Naja naja oxiana reduces the activity to about 20 ~ (to be published). It has already been mentioned that in the long neurotoxin siamensis 3, the charge of the 8-amino group of lysine-53 can be removed with retention of 67 ~ of the toxicity. Furthermore, toxin I (structural type 73-5) of Naja raja oxiana has a glutamic acid residue in position 53. Iodination oferabutoxin b (type 62-4) of Laticauda semifasciata and the formation of düodohistidine at position 26 had no effect on the toxicity (SATO and TAMIYA, 1970). The cystine residues do not take part in any disulfide interchange reactions with the acetylcholine receptor . The `extra' disulfide 2a (Fig. 5) of toxin a of Naja nivea can be reduced and alkylated with retention of antigenicity and 9 ~ of the toxicity (Booms, 1974) . The purified acetylcholine receptor can be treated with a 1600-fold molar excess of iodoacetate to alkylate any possible SH-groups without abolishing the neurotoxin binding capacity (Knxt.ssoN et al., 1976) . Modification with phenylglyoxal of the invariant arginine-37 together with arginineTOXlCON 1976 Yo1. 74

Tyrosine Nitration in Cobra Neurotoxins

f 7=Y~iü :Ygl d~ü y~ :XYY$$ = : :» » 9q â i > T »Y ~ i » f > ~ ~ ~ ~ ~ YY7GdYILIL W ii~~L~

S1 Qf

;Fi~FrrFf~FfF~ff-f-F ~uama , .~ :iâ

7~OXICON 1976 Yol. 14

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EVERT KARLSSON and JOHAN SUNDELIN

28 in cobrotoxin gave a derivative with 25 ~ residual activity (Ynxc et al., 1974). The reaction with phenylglyoxal is, however, slowly reversible at neutral pH (TAKAHASHI, 1968), and one can therefore not exclude the possibility that the residual toxicity might have been due to a reversal of the reaction prior to assay or in vivo. The binding between the toxin and its target, the acetylcholine receptor, is very strong and is probably the result of the interaction between several groups in the toxin and the receptor . It seems probable that at least one amino, carboxyl, tyrosyl, or tryptophanyl group should be in contact with the receptor in the toxin-receptor complex. If so, it should thus be possible to modify such an interacting group without inactivating the toxin. The interaction from the remaining groups should still be sufficient to bind the toxin to the receptor. The effect of such a modification might only be a less strong binding, which should be manifested as a lower toxicity. Chemical modifications as done so far might therefore not give any information about amino acid residues interacting with the receptor . We think that another approach is necessary. The experiments have rather to be done with the receptor-toxin complex and aim at identifying amino acids accessible to reagents. The non-accessible ones (if normally accessible) should be in the interior of the complex, presumably in contact with the receptor. A toxin molecule must have characteristic features serving as a recognition site for the receptor, otherwise the localization of the target should not be possible. There exists a great variety of cholinergic drugs. The structures of substances such as acetylcholine, decamethonium, phenyltrimethylammonium, n-tubocurarine, and the venom neurotoxins have little in common except a strongly positively charged group. We think that this group serves as the recognition site. A modification involving a removal of the charge should then abolish the activity as the ability to recognize the target should be lost. The neurotoxins have two invariant cationic groups ; the N-terminal amino group which can be modified with retention ofthe activity (KAxr ssox et a1.,1972) and the guanidino group of arginine-37, the role ofwhich is still uncertain due to the limitations of the modifying reaction . We think that arginine-37 provides the postulated recognition site. Acknowledgements-We thank Dr. Dwro Era for the gift of the Ngja nigricollis toxin a and for the amino acid analyses . This work was supported by a grant (dnr 2859-8) from the Swedish Natural Science Research Council . REFERENCES

Axrn3Etta, H., Eax>:at, D., FRYKLUND, L. and I{~xis~ox, E. (1974) Amino acid sequence of oxiana a, the main neurotoxin of the venom of Naja raja oxiana. Biochem. biophys. Acts 359, 222. B~u~ttcs, B. B. C., Mu sni, R. and Stm'or nJl, R. A. (1974) The primary sequences and neuromuscular effects of three neurotoxic polypeptides from the venom of Dendroaspls viridis. Eur. J. Biochem. 45, 457. BozFS, D. P. (1971) The amino acid sequences of toxins a and ß from Naja nivea venom and the disulfide bonds of toxin a. J. biol. Chem. 246, 7383 . Borns, D. P. (1972) The amino acid sequences of toxins b and dfrom Naja melanoleuca venom. J. blot. Chem . 247, 2866. BorFS, D. P. (1974) Snake venom toxins . The reactivity of the disulphide bonds of Naja nivea toxin a. Biochim. biophys. Acts 359, 242. BoT6s, D. P. and SrxvnoM, D. J. (1969) A neurotoxin, toxina from Egyptian cobra (Ngja lraje hale) venom. I. Purification, properties, and complete amino acid sequence. J. blot. Chem. 244, 4147. Bores, D. P., SrxvnoM, D. J., AxnEnsox, C. G. and G~IxISreNSenr, P. A. (1971) Snake venom toxins . Purification and properties of three toxins from Naja nivea (Linnaeus) (Cape cobra) venom and the amino acid sequence of toxin S. J. blol . Chem . 246, 3132 . CHnxa, C. C. and Yaxa, C. C. (1973) Immunochemical studies on the tryptophan modified cobrotoxin . Blochlm. biophys. Acra 295, 595. CHANO, C. C., Y.~xa, C. C., HAMAaUCHi, K., N~ICwi, K. and Hnv.4sFlt, K. (1971a) Studies on the status of tyrosyl residues in wbrotoxin. Blochim. biophys. Acts 236, 164. TOXICON 1976 Vol. If

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Cawxe, C. C., YwNG, C. C., Nwgwi, K. and HAYA~II, K. (1971b) Studies on the status of free amino and carboxyl groups in cobrotoxin. Blochlm. biophys. Acts 251, 334. Cfncm3roazccm?, R., Roc13wT, C., Swt~tu, F. and Lwznurrsss, M. (1972) Structure-function relationships of neurotoxins isolated from N~a hgje venom. Physioochariical properties and idwtification of the alive site. Biochemistry 11, 1681 . C~rwzxac .~ses, P., Fucxs, S. and Axrn~serr, C. B. (1968) The tyrosyl residues at the alive site of staphylococcal nuclease. Modifications by tetranitromethane. J. btal. Chem. 243, 4787 . EwxEe, D. and POrtwTx, J. (1967) The amino acid sequence of a neurotoxin from N~a nigrlcollis venom. 7th Int. Cong. Biochem., Tokyo, 1967, Col. VIII-3, Abstracts III, p. 499. Tokyo: The Science Council of Japan. fhvxt urm, L., EAKER, D. and Kwarssox, E. (1972) Amino acid sequences of the two principal neurotoxins of EnhydrinaschLstosa venom. Biochemistry 11, 4633 . Gxi~x, E. V., Suxmxx, A. P., Lumrwxcsux, N. N., Si oeonvwx, L. N., Lmtrnv, V. M., Ovcfmvrm

Nitration of tyrosine in three cobra neurotoxins.

Toxtcow, 1976, Vo4 14, pp. 295-306. ParQamon Prey . Printed in C3reat Britain. NITRATION OF TYROSINE IN THREE COBRA NEUROTOXINS EVERT 11AAL 7AlJN and...
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