Dawie P. Botes

The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the cyanobacterium Microcystis aeruginosa

The structure of cyanoginosin-LA (previously referred to by us as toxin BE-4) is cyclo(-D-Ala-L-Leu-erythro-β-methyl-D-isoAsp-L-Ala-Adda-D-Glu-N-methyldehydroAla)(17a), where Adda refers to the novel β-amino acid residue of 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (11). The connectivity of Adda is deduced from 1H- and 13C-n.m.r. data, but the stereochemistry at carbons 2,3,8, and 9 remains unknown. The residue sequence is derived from the mass spectra of cyclic and linear derivatives of the toxin. A new nomenclature for the related toxins of Microcystis aeruginosa is discussed.

Structural studies on cyanoginosins-LR, -YR, -YA, and -YM, peptide toxins from Microcystis aeruginosa

The structures of the hepatotoxins of general name cyanoginosins-XY are proposed to be cyclo-D-Ala-L-X-erythro-β-methyl-D-isoAsp-L-Y-Adda-D-isoGlu-N-methyldehydroAla (1) where X and Y represent variable amino acids and Adda is 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (2). The structural studies on four variant toxins utilised n.m.r. and mass spectral methods analogous to those recently used to determine the structure of cyanoginosin-LA. [graphic omitted] (1)

Isolation and characterization of four toxins from the blue-green alga, Microcystis aeruginosa

Two alternative procedures for the isolation of toxins from the blue-green alga, Microcystis aeruginosa forma aeruginosa, are described. A novel approach is reported, whereby contaminating impurities are succinylated, exploiting the absence of free amino groups in toxin variants. All toxin variants comprise a hydrocarbon blocking group, five amino acid residues detectable by conventional means, while methylamine is liberated upon acid hydrolysis. Possible structural features are discussed relating to the observed chemical and physical properties of the toxins.

A new type of toxin in the venom of snakes of the genus Atractaspis (Atractaspidinae).

The venom of Atractaspis is unique in having a large percentage of both high and low molecular weight components. Its Sephadex G-50 S5 fraction appears to represent a new type of toxin that contains 17-18 Asx, 13-14 Cys and 10-11 Glx out of a total of 72-78 amino acids. The N-terminal of this toxin does not seem to resemble any of the known toxins. The overall lethal potency of the venom is very high; i.v. injections of 5 microgram of venom or 1 microgram of fractions S5 or S6 per mouse causes death within minutes. The results of the present study corroborate previous findings that suggested a separate grouping of the snakes genus Atractaspis at the subfamilial or familial level.

Configuration assignments of the amino acid residues and the presence of N-methyldehydroalanine in toxins from the blue-green alga, Microcystis aeruginosa

The configuration assignment of the alpha-carbon atom of amino acid residues in four toxin variants from Microcystis aeruginosa have been made by stereospecific enzymic transformations. The relative conformation assignment of the beta-carbon atom of beta-CH3-aspartic acid could be made by comparison of the electrophoretic mobility with literature values reported for the authentic compound. The presence of an N-methyldehydroalanine residue, which, due to elimination of methylamine under hydrolytic conditions, previously escaped detection by conventional means, has been confirmed by identification of N-methylalanine in the hydrolysate after reduction of toxin with sodium borohydride.

Monoclonal antibody specific for cyanoginosin-LA: Preparation and characterization

The toxin cyanoginosin-LA (MW 909), isolated from Microcystis aeruginosa, was successfully conjugated with polylysine and muramyl dipeptide to form a high molecular weight complex consisting of a hapten, a carrier and a built-in adjuvant. This complex was used for the immunization of mice. Monoclonal antibodies specific for cyanoginosin-LA were produced using the hybridoma technique. The ten most efficient producers of these antibodies were further characterized and the monoclonal antibody produced by them was found to be identical on the basis of additivity test, isoelectric focussing and sub-class immunoglobulin typing results. The anti-cyanoginosin-LA monoclonal antibody focussed at a pH of approximately 6.55 and was found to belong to the mouse immunoglobulin subclass IgM. Large scale production of the antibody (in vivo) was followed by purification with ammonium sulphate precipitation and high performance liquid chromatography. The anticyanoginosin-LA monoclonal antibody, when reacted with six variants of cyanoginosin (other than cyanoginosin-LA), bound all with equal efficiency.

Separation of Bitis gabonica (Gaboon adder) venom arginine esterases into kinin-releasing, clotting and fibrinolytic factors

Abstract C. C. Viljoen , C. M. Meehan and D. P. Botes . Separation of Bitis gabonica (Gaboon adder) venom arginine esterases into kinin-releasing, clotting and fibrinolytic factors. Toxicon17, 145–154, 1979. The arginine ester hydrolysing activity of the venom of Bitis gabonica has been isolated and purified by a combination of Sephadex gel filtration and ion-exchange chromatography on DEAE-cellulose. The esterases were designated E-I, E-II and E-III according to their sequence of elution from DEAE-cellulose. A molecular weight of about 32,000 was calculated for the enzymes which contain 20–25% by weight of carbohydrate. The enzymes appear to be specific for arginine esters, since lysine esters are not hydrolysed, and showed low proteolytic activity against casein. Distinct biological properties were found for the esterases, E-I, EII and E-III showing kinin-releasing, fibrinolytic and clotting activity, respectively.

Isolation and amino acid sequence of caudoxin, a presynaptic acting toxic phospholipase A2 from the venom of the horned puff adder (Bitis caudalis)

A presynaptic acting toxic phospholipase A2, designated caudoxin, was purified from the venom of Bitis caudalis by a combination of gel filtration and ion-exchange chromatography. The specificity of the enzyme was shown to be of the A2 type. The enzyme contains 121 amino acid residues in a single chain and is cross-linked by seven disulfide bridges. Application of cyanogen bromide cleavage and digestion with trypsin and chymotrypsin yielded peptides providing the necessary overlaps to complete derivation of the sequence. Structural features of caudoxin in relation to other toxic and non-toxic phospholipases A2 are discussed.

Purification of phospholipase A from Bitis gabonica venom

Abstract Bitis gabonica venom was separated on Sephadex G-50 and the major enzymes present in the venom localized by assaying the eluate against specific substrates. Phospholipase A was purified and its positional specificity towards phosphatidylcholine shown to be of the A 2 type. The amino acid composition of the purified phospholipase A is reported. An equilibrium is shown to exist between monomer and dimer which is favoured towards the dimer by addition of Ca 2+ and towards the monomer by EDTE.

Snake venom toxins—I. Preliminary studies on the separation of toxins of elapidae venoms

Abstract The venoms of nine Elapidae snakes representing three genera were fractionated by chromatography on Amberlite CG-50 and Sephadex G-50 and the elution positions of their toxins determined. Two or more distinct toxins were contained in each of these venoms. A very low molecular weight toxic component was found in Naja naja and two of the three Dendroaspis venoms. Its molecular weight is estimated at 3000–4000, i.e. nearly half the size of the toxins usually found in front fanged snakes. Dendroaspis angusticeps venom contained only biologically deleterious components which failed to kill mice although the whole venom is highly toxic. It is possible that a combination of fractions from this venom is necessary for its lethal action.