D.D. Sheumack

Amino acid analysis using derivatisation with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography

A simple procedure for the precolumn derivatisation of amino acids with 9-fluorenylmethyl chloroformate and a liquid chromatographic method for the separation of the derivatives with fluorimetric detection in the picomole range are reported. The procedure does not involve a solvent extraction and gives single, stable derivatives of the common protein amino acids. The method has been demonstrated on hydrolysates of proteins and peptides.

Complete amino acid sequence of a new type of lethal neurotoxin from the venom of the funnel-web spider Atrax robustus

Robustoxin, the lethal neurotoxin isolated from the venom of the male Sydney funnel–web spider, Atrax robustus, is of unique structural type and physiological mode of action. The primary structure of this 42–residue peptide was determined to be H2N–Cys–Ala–Lys–Lys–Arg–Asn–Trp–Cys–Gly–Lys10–Asn–Glu–Asp–Cys–Cys–Cys–Pro–Met–Lys–Cys20–Ile–Tyr–Ala–Trp–Tyr–Ala–Trp–Tyr–Asn–Gln–Gln–Gly–Ser30–Cys–Gln–Thr–Thr–Ile–Thr–Gly–Leu–Phe–Lys40–Lys–Lys–Cys–H The disposition of disulphide–bridged cysteine residues at both the amino– and carboxy–termini and as a triplet at residues 14–16 appears to have no precedent amongst neurotoxins.

Applications of automated amino acid analysis using 9-fluorenylmethyl chloroformate

A rapid and sensitive fully automated method for the determination of primary and secondary amino acids in different matrices is described. Amino acids are derivatized with 9-fluorenylmethyl chloroformate using an automated precolumn derivatization technique. Data are presented to show that the technique is both reproducible and highly sensitive. Applications of the technique are presented, including the analysis of peptide and protein hydrolysates and the profiling of free amino acids in physiological fluids.

Improved high-performance liquid chromatography of amino acids derivatised with 9-fluorenylmethyl chloroformate

Abstract An improved high-performance liquid chromatography method for the separation of amino acids derivatised with 9-fluorenylmethyl chloroformate (Fmoc) is described. This method, in conjunction with a multi-tasking program, not only allows high automatic througput but also offers baseline resolution of the common Fmoc-amino acids from a linear acetonitrile gradient, greater chromatographic reproducibility over the life of the column (800 runs) and easy column regeneration. The methods described are shown to be suitable for analysis of hydrolysates of proteins from two-dimensional gels for protein identification purposes, and will also be useful for routine quality control and screening of biological samples.

Actions of robustoxin, a neurotoxic polypeptide from the venom of the male funnel-web spider (Atrax robustus), in anaesthetized monkeys

Robustoxin, a polypeptide consisting of a chain of 42 amino acid residues in a known sequence, has been isolated by cation exchange chromatography from the crude venom of the male funnel-web spider (Atrax robustus). Physiological activity or toxicity in the venom fractions was detected by production of fasciculation in mouse phrenic nerve-hemidiaphragm preparations and by lethality in new-born mice. In the present experiments in Macaca fascicularis monkeys anaesthetized with pentobarbitone, robustoxin (5-30 micrograms/kg infused i.v. over 5 min) produced immediate disturbances in respiration (including dyspnoea and apnoea), blood pressure and heart rate followed by severe hypotension (mean systemic blood pressure less than 50 mmHg) or death due to respiratory and circulatory failure within 196 min. Robustoxin also produced lachrymation, salivation, generalized skeletal muscle fasciculation and a parallel increase in body temperature, and increased firing in skeletal motor and autonomic nerves. These effects closely resembled those produced by i.v. infusions over 5 min of 50 micrograms/kg of crude venom from male A. robustus spiders. Crude venom from female A. robustus spiders (500 micrograms/kg i.v. over 5 min) produced some of the effects elicited by robustoxin and crude venom from male spiders, but to a much less marked extent. It was concluded that robustoxin is responsible for the neurotoxic and lethal effects of human envenomation by male A. robustus spiders.

A comparative study of properties and toxic constituents of funnel web spider (Atrax) venoms.

The crude venoms of male and female Sydney funnel web spiders, Atrax robustus, were compared by cation-exchange and high-performance liquid chromatography, lethality to new-born mice, polyacrylamide gel isoelectric focusing, immunoelectrophoresis, phospholipase A analysis, effects on the mouse phrenic nerve hemidiaphragm and passive paw oedema in the rat and, except in the case of rat paw oedema, were found to exhibit quite different properties. Polyacrylamide gel isoelectric focusing and high-performance liquid chromatography proved to be suitable for gender and species determination when applied to the venoms of A. formidabilis, A. infensus, A. robustus and A. versutus. These venoms were also compared by lethality, promotion of muscle fasciculation and phospholipase A activity.

Occurrence of a tetrodotoxin-like compound in the eggs of the venomous blue-ringed octopus (Hapalochlaena maculosa).

A lethal toxin was isolated and partly purified from the eggs of the blue-ringed octopus, Hapalochlaena maculosa. Examination of the toxin by thin layer chromatography, isoelectric focusing and its effects upon the compound nerve action potentials of the toad sciatic nerve gave results that were indistinguishable from those displayed by authentic tetrodotoxin, the toxin present in the venom glands of the octopus.

The complete amino acid sequence of a post-synaptic neurotoxin isolated from the venom of the Australian death adder snake Acanthophis antarcticus.

1. A lethal neurotoxin (acanthophin d) was isolated from the venom of the Australian death adder snake Acanthophis antarcticus. 2. Acanthophin d consisted of a single polypeptide chain of 74 amino acid residues cross-linked by five disulphide bridges. 3. The results of neurophysiological experiments on murine phrenic nerve hemi-diaphragm preparations were consistent with irreversible post-synaptic blockage of neuromuscular transmission by acanthophin d.

Isolation and partial characterisation of a lethal neurotoxin from the venom of the Australian death adder (Acanthophis antarcticus).

Abstract The venom of the Australian terrestrial snake Acanthophis antarcticus was subjected to gel permeation and ion exchange chromatography and to isoelectric focusing, and five different lethal fractions were separated. The application of preparative isoelectric focusing to one of these fractions yielded a lethal peptide toxin named acanthophin a, which has been partially characterised. Our experiments confirm earlier work that the crude venom causes death by blocking neuromuscular transmission. Acanthophin a also has this action. It consists of a single chain of 63 amino acid residues crosslinked by four disulphide bridges. It has an approximate i.p. ld 50 value in mice of 0·16 mg/kg body weight.

Protection of monkeys against the lethal effects of male funnel-web spider (Atrax robustus) venom by immunization with a toxoid

A stable toxoid was prepared from robustoxin (the lethal polypeptide neurotoxin in the venom of the male funnel-web spider, Atrax robustus) by polymerization with glutaraldehyde. This material was non-toxic in new-born mice. Administration of the toxoid to three Macaca fascicularis monkeys (50-80 micrograms/kg s.c. at 14-day intervals for 8-12 weeks) produced no toxic effects; anti-robustoxin antibodies were detected in serum samples by immunodiffusion tests within 13-27 days. In vivo evidence of successful protection with the toxoid was obtained by challenging the monkeys with male A. robustus venom (50 micrograms/kg i.v.) under anaesthesia with pentobarbitone (one monkey), or with ketamine, halothane and nitrous oxide, 1-26 weeks after the last injection of the toxoid. Only minor respiratory, cardiovascular and skeletal motor disturbances were produced, and all monkeys recovered fully and uneventfully. Challenge with the same dose of venom in non-immunized or robustoxin N-terminal decapeptide ovalbumin conjugate-treated monkeys resulted in typical lethal neurotoxic effects, culminating in severe hypotension or death from circulatory and respiratory failure within 280 min.