Fritz H.H. Carlsson

Snake venom toxins The isolation and purification of three cytotoxin homologues from the venom of the forest cobra (Naja melanoleuca) and the complete amino acid sequence of toxin VII1

Three proteins (VII1–VII3) have been isolated from the venom of Naja melanoleuca (Forest cobra). The complete amino acid sequence of VII1 has been established. The sequence of VII1 and the amino acid composition and other data for VII2 and VII3 indicate that the three proteins are homologoes of the cyto- or cardiotoxins which have been isolated from the venoms of other members of the Elapidae sub-family. Protein VII1 showed the intravenous toxicity level typical of cytotoxin type proteins, while VII2 and VII3 were considerably less toxic. The sequence of the first 34 residues, out of the total of 60, was determined using an automatic sequenator. The remaining 26 residues were aligned with the aid of tryptic and chymotryptic peptides and thermolysin produced sub-fragments of one tryptic peptide. It was found that VII1 consisted of a pair of isoproteins having leucine and isoleucine NH2-terminal residues.

Snake venom toxins the primary structure of protein S4C11: A neurotoxin homologue from the venom of forest cobra (Naja melanoleuca)

Six minor protein constituents (S4C10-S4C15) have been isolated from the venom of Naja melanoleuca. The complete amino acid sequence of S4C11 has been established and indicates that it is a homologue of the neurotoxins which are found in elapid venoms. The other proteins appear from the amino acid compositions to be homologues of the cyto- or cardiotoxins found in cobra venoms. Protein S4C11 has a low toxicity, failing to kill mice at an intravenous dose of 20 mug/g body weight. The sequence of the first 25 residues out of the total of 65, was determined using the automatic sequenator. The remainder of the sequence was derived with the aid of tryptic and chymotryptic peptides. The sequence showed the unusual feature of having 65 amino acid residues including 10 half-cystine residues.

Snake venom toxins the primary structures of two novel cytotoxin homologues from the venom of forest cobra (Naja melanoleuca)

Summary The primary structures of V II 2 and V II 3, two closely related basic proteins of low toxicity from Naja melanoleuca venom are described. Both consist of single 61-residue chains cross-linked intramolecularly by four disulphide bridges. They differ by a lysine-asparagine substitution at position 36, V II 3 being the more basic. A high degree of homology with the cytotoxin (cardiotoxin) group is evident. The unusual features of the sequences when compared to the cytotoxins are, the radical substitutions at positions 19, 25, 34, 36 and 41 and particularly the sequence of residues 38–44 i.e. Gly-Cys-Ala-Ala-Thr-Cys-Pro which occurs at the corresponding position in all the 71–74 (long) neurotoxins. The possible implication of certain amino acid substitutions is discussed.

The preparation of 3-nitrotyrosyl derivatives of three elapid venom cardiotoxins.

Nitration studies using tetranitromethane were conducted on the tyrosine residues of cardiotoxins, naja melanoleuca VII1, Naja haje annulifera VII1 and Hemachatus haemachates toxin 12B. Various partially and fully nitrated derivatives were formed. Analysis of the products of nitrating naja melanoleuca VII1 showed that the average relative reactivities of the three tyrosine residues were Tyr-25 greater than Tyr-22 greater than Tyr-51. It was significant that Tyr-51 could be easily modified in both N. melanoleuca VII1 and N. haje annulifera VII1. In contrast, other workers had found Tyr-51 in N. naja atra cardiotoxin to be unreactive towards tetranitromethane except under denaturing conditions. Fully nitrated derivatives of N. melanoleuca VII1 (Tyr-22, -25 and -51), N haje annulifera VII1 (Tyr-22 and -51) and H. haemachates 12B (Tyr-22), prepared under mild reaction conditions, were isolated by ion-exchange and hydrophobic interaction chromatographies. All three derivatives were pure by disc gel electrophoresis at pH 8.9 and amino acid analysis. They were therefore suitable for spectral and biological studies. The results were compared and contrasted to; those of other workers.

The selective S-alkylation of a methionine residue in an elapid venom cardiotoxin

1. The reaction of cardiotoxin with iodoacetamide or iodomethane at pH 3.0 afforded the corresponding methionine sulphonium derivatives. The major products were S-alkylated at Met-26 whilst the minor products were S-alkylated at both Met-24 and -26. 2. Reaction with iodoacetamide under denaturing conditions led to a reversal of the relative abundances of the two derivatives in the respective reaction mixtures. 3. The derivative S-methylated at Met-26 was about 5-fold less toxic than the parent cardiotoxin. That derivatised at both Met-24 and -26 was non-toxic, indicating the importance of Met-24. 4. The results are discussed in the light of a structural model, previous chemical modifications and 1H-NMR data. It appeared that Met-24 is important for the integrity of an important structural feature of cardiotoxin.

Properties of some 3-nitrotyrosyl elapid venom cardiotoxins.

Nitration of the invariant Tyr-22 in Hemachatus haemachates cardiotoxin 12B did not greatly decrease lethality, and the haemolytic potency towards guinea-pig erythrocytes remained unchanged. This residue is thus non-essential for cardiotoxin to exert its biological action. Nitration of Naja haje annulifera and Naja melanoleuca cardiotoxins VII1 decreased but did not abolish the lethalities and haemolytic potencies. Thus Tyr-25 and Tyr-51 were concluded to have no direct functional role in cardiotoxin lethality. The pKa values of the phenolic hydroxyl groups of the tyrosine residues appeared to be important for certain properties of cardiotoxin in solution. No evidence could be produced to show that Tyr-51 is unreactive to nitration under normal (non-denaturing) conditions.

The modification of arginine-36 in elapid venom cardiotoxin using 1,2-cyclohexanedione

Abstract The importance of the invariant arginine-36 in cardiotoxin was investigated by chemical modification. Reaction of the single arginine residue in Naja haje annulifera venom cardiotoxin V 11 1 with 1,2-cyclohexanedione yielded derivative 1 (cardiotoxin V 11 1 modified at Arg-36). Purification was achieved using cation-exchange chromatography in the presence of borate ions. The reaction was reversible by incubation with hydroxylamine at 37°C. Derivative 1 had an intravenous LD 50 of 17 μg·g −1 in mice, the borate complex of the derivative an LD 50 of 14 gmg·g −1 , and the maternal resulting from the reversal of the reaction, 5.9 μg·g −1 , as compared to an LD 50 of 4.5 μg·g −1 for the native toxin. Circular dichroism spectra in borate buffer (pH 8.0) and glycine-HCl (pH 3.5) showed that the derivative had not undergone severe conformational changes. Because some lethality was retained after making the derivative, it is doubtful whether Arg-36 can be considered to be of crucial importance for the biological action of cardiotoxin and it probably fulfils a structural role.