ABSTRACT
Robustoxin, the lethal neurotoxin from the Sydney funnel web spider Atrax robustus, is a polypeptide of 42 residues cross-linked by four disulfide bonds. This paper describes the sequence-specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of robustoxin in aqueous solution. Several broad backbone amide resonances were encountered in spectra recorded at 27 degrees C, making the assignments at that temperature incomplete. In spectra recorded at lower temperatures these amide resonances became sharper, but others that were sharp at 27 degrees C became broad, indicative of conformational averaging on the millisecond timescale for certain regions of the structure. Nevertheless, it was possible to establish that robustoxin contains a small, triple-stranded, antiparallel beta-sheet and several reverse turns, but no alpha-helix. These observations indicate that this toxin may adopt the inhibitor cystine knot structure found in polypeptides from a diverse range of species, including a number of spiders. Analysis of the pH dependence of the spectrum yielded pKa values for Tyr22 and Tyr25, one of the three carboxyl groups, and the Lys residues.
Subject(s)
Neurotoxins/chemistry , Peptides/chemistry , Spider Venoms/chemistry , Spiders/metabolism , Amino Acid Sequence , Animals , Hydrogen , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Structure, SecondaryABSTRACT
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.
Subject(s)
Amino Acids/analysis , Autoanalysis/methods , Chromatography, High Pressure Liquid/methods , Fluorenes , Adult , Amino Acid Metabolism, Inborn Errors/blood , Amino Acids/blood , Child, Preschool , Drug Stability , Humans , Indicators and Reagents , Male , Microchemistry , Phenylketonurias/blood , Quality Control , Tyrosine/blood , Tyrosine Transaminase/deficiencyABSTRACT
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.
Subject(s)
Amino Acids/analysis , Angiotensin II/chemistry , Chromatography, High Pressure Liquid/methods , Fluorenes , Humans , Microchemistry , Muramidase/chemistryABSTRACT
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.
Subject(s)
Neurotoxins/immunology , Spider Venoms/immunology , Toxoids/immunology , Amino Acid Sequence , Animals , Animals, Newborn/physiology , Blood Pressure/drug effects , Glutaral , Heart Rate/drug effects , Immunodiffusion , Macaca fascicularis , Mice , Molecular Sequence Data , Neurotoxins/toxicity , Peptide Fragments/immunology , Salivation/drug effects , Spider Venoms/toxicity , Tears/metabolismABSTRACT
1. An endogenous antitoxin fraction was isolated from non-immune rabbit sera by affinity chromatography with robustoxin bound to the solid support. 2. Robustoxin is the sole lethal toxin in the venom of the male funnel web spider, Atrax robustus. 3. The fraction was found to contain IgG and IgM immunoglobulins. 4. This fraction prevented or reversed the lethal actions of the crude venom in newborn mice, in mouse phrenic nerve-hemidiaphragm preparations, and in anaesthetized monkeys. 5. The antitoxin fraction is of potential value in the therapy of human envenomation by A. robustus.
Subject(s)
Antitoxins/isolation & purification , Spider Venoms , Animals , Animals, Newborn , Antitoxins/pharmacology , Chromatography, Affinity , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Macaca fascicularis , Male , Mice , Rabbits , Spider Venoms/toxicityABSTRACT
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.
Subject(s)
Elapid Venoms , Neurotoxins , Amino Acid Sequence , Amino Acids/analysis , Animals , Chemical Fractionation , Chymotrypsin , Elapid Venoms/isolation & purification , Elapid Venoms/pharmacology , In Vitro Techniques , Mice , Molecular Sequence Data , Neuromuscular Junction/drug effects , Neurotoxins/isolation & purification , Peptides/analysis , Synaptic Transmission/drug effects , TrypsinABSTRACT
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.
Subject(s)
Arthropod Venoms/toxicity , Spider Venoms/toxicity , Anesthesia , Animals , Blood Pressure/drug effects , Crying/physiology , Electromyography , Female , Heart Rate/drug effects , Injections, Intramuscular , Injections, Intravenous , Macaca fascicularis , Male , Salivation/drug effectsABSTRACT
A method for obtaining total protein patterns from lipid-containing systems, in particular egg yolk, is described. After dispersion of the yolk in 8 M guanidine hydrochloride solution, lipid is removed by extraction with chloroform-methanol and petrol. The protein solution is applied to a high-pressure liquid chromatograph and eluted with a gradient of formic acid, isopropanol, and acetonitrile. In measurements on a known yolk protein, duck apovitellenin I, the method did not cause irreversible formylation of N-terminal or other residues. The method was used (1) to compare protein patterns of whole yolk from hen and quail eggs; (2) to isolate and partially sequence quail apovitellenin I; and (3) to compare protein patterns of "white yolk" and "yellow yolk."
Subject(s)
Egg Proteins/isolation & purification , Animals , Chickens , Chromatography, High Pressure Liquid/methods , Coturnix , Eggs , Electrophoresis, Polyacrylamide Gel/methods , Female , Formates , Lipids , SolventsABSTRACT
The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.
Subject(s)
Arthropod Venoms/analysis , Spider Venoms/analysis , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Female , Male , Peptide Fragments/analysisABSTRACT
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-Lys-Asn-Glu-Asp-Cys-Cys-Cys-Pro- Met-Lys-Cys-Ile-Tyr-Ala-Trp-Tyr-Asn-Gln-Gln-Gly-Ser-Cys-Gln-Thr-Thr-Ile- Thr-Gly-Leu-Phe-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.
Subject(s)
Arthropod Venoms/analysis , Spider Venoms/analysis , Amino Acid Sequence , Animals , MaleABSTRACT
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.
Subject(s)
Arthropod Venoms/toxicity , Spider Venoms/toxicity , Animals , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Exocrine Glands/metabolism , Female , Immunoelectrophoresis , Inflammation/chemically induced , Isoelectric Focusing , Lethal Dose 50 , Male , Mice , Muscle Contraction/drug effects , Phospholipases A/metabolism , Rats , Spider Venoms/analysis , Spider Venoms/immunology , SpidersABSTRACT
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.
Subject(s)
Mollusk Venoms/toxicity , Ovum/analysis , Tetrodotoxin/toxicity , Action Potentials/drug effects , Animals , Bufo marinus , Female , In Vitro Techniques , Octopodiformes/physiology , Sciatic Nerve/drug effectsABSTRACT
Maculotoxin, a potent neurotoxin isolated from the posterior salivary glands of the blue-ringed octopus. Hapalochlaena maculosa, has now been identified as tetrodotoxin. This is the first reported case in which tetrodotoxin has been found to occur in a venom.
