Donald R. Babin

Amino acid sequences of neurotoxic protein variants from the venom of Centruroides sculpturatus Ewing

Abstract The venom from the scorpion, Centruroides sculpturatus Ewing (range, Southwestern United States), was fractionated into ten protein zones by chromatography on CM-cellulose. Further purification of three of these zones on DEAE Sephadex in ammonium acetate developers yielded three principal neurotoxins designated variants 1, 2, and 3. Variants 1 and 3 each consist of 65 amino acid residues, variant 2 is composed of 66 residues, and each variant is a single polypeptide chain crosslinked by four disulfide bridges. The three variants have lysine at the amino terminus, serine at the carboxyl terminus, and their sequences exhibit a high degree of homology. The complete structure of variant 2 was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. The sequences of most of the tryptic peptides of variants 1 and 3 were determined, and the peptides were aligned by comparison with the homologous peptides in variant 2. The results show that there are 4 differences in sequence between variants 2 and 3 and 9 differences between variants 1 and 2. Variants 1 and 3 differ from each other at 5 positions in their sequences. These differences between the three protein variants are found in the amino terminal one-third of the molecules except for the deletion of one seryl residue at the carboxyl terminal of variants 1 and 3.

Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue. The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions. The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: The primary structure☆☆☆

The complete primary structure of five chymotrypsin/elastase isoinhibitors isolated from Ascaris lumbricoides was determined by conventional methods. These structures represent the first sequence set for the Ascaris inhibitor family. All five isoinhibitors are single-chain polypeptides crosslinked by five disulfide bridges. Isoinhibitor 1 consists of 63 amino acid residues and has glycine at the N-terminal and histidine at the C-terminal. Isoinhibitors 2-5 all have arginine at the N-terminal, differ at positions 25 and 40, and have different C-terminal regions. Isoinhibitors 2 and 4 have asparagine at positions 25 and serine at position 40, whereas isoinhibitors 3 and 5 have lysine and threonine at these positions, respectively. The different C-terminal regions of isoinhibitors 2-5 account for their varying lengths. Isoinhibitor 1 has no sequence heterogeneity. Frequent repetitions of various dipeptides and one tripeptide are evident along the peptide chain of isoinhibitors 2-5. None of the isoinhibitors contains the aromatic amino acids phenylalanine or tyrosine. Comparison of the amino acid sequence of isoinhibitor 1 with the sequence of isoinhibitors 2-5 shows that they differ at a minimum of 16 positions. The primary structures of isoinhibitors 1-5 from Ascaris do not demonstrate a great degree of homology when compared with the sequence of presently known proteinase inhibitors. However, these isoinhibitors share with a very large number of inhibitor families the presence of half-cystine in the P3 position.

Allergens in bee venom: II. Two new high molecular weight allergenic specificities

Two new allergenic specificites were detected in honeybee venom and the two corresponding protein substances isolated by gel filtration, immunoadsorption, and ion exchange chromatography. The first of these, allergen B, has a molecular weight ranging from 49,000 to more than 200,00 d and can be recognized by rabbit and guinea pig antisera as well as by human reaginic sera using the radioallergosorbent test (RAST). Allergen B gives a single line in immunodiffusion distinct from hyaluronidase, phospholipase A, melittin, and the other high molecular weight substances described and gives a single band at 49,000 d in sodium dodecyl sulfate (SDS) polyacrylamide gel. The second substance, allergen C, has a molecular weight of 105,000 d and was separated from allergen B by immunoadsorption with insoluble antibody. Allergen C was shown to be distinct from the other sustances in bee venom by immunodiffusion with animal antisera. One human reaginic serum was monospecific for allergen C. Two other minor components of 86,000 and 71,000 d are present in bee venom; their allergenic activities are unknown. The two specifities, B and C, comprise most of the reactivity of the previously described Sephadex G-75 fraction 1 and clearly are important allergens, reacting with 98% of sera from bee venom-allergic individuals.

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: isolation by affinity chromatography and association with the enzymes.

Five isoinhibitors, proteins that inactivate chymotrypsin and elastase, were isolated from aqueous extracts of the intestinal parasite Ascaris lumbricoides var. suum by affinity chromatography. They were named in the order that they eluted from a CM-Sephadex C-25 column at pH 8.6 using a salt gradient. Isoinhibitor 1, first reported in this paper, is anionic on polyacrylamide gel electrophoresis at pH 9.3. The other four isoinhibitors are cationic on electrophoresis at pH 9.3, separable from each other, and identical with those reported previously [R.J. Peanasky and G. M. Abu-Erreish (1971) in Proceedings International Research Conference on Proteinase Inhibitors (Fritz, H., and Tschesche, H., eds.), pp. 281-293, de Gruyter, New York]. Amino acid compositions show differences between the isoinhibitors. Antibody to isoinhibitor 1 reacts with its self-antigen only. Antibody to isoinhibitor 5 reacts with isoinhibitors 2-5 but not with isoinhibitor 1. Association equilibrium constants show that each of the isoinhibitors interacts most avidly with alpha-chymotrypsin. For isoinhibitor 1, the K alpha for alpha-chymotrypsin was 2.6 X 10(11) M-1, for porcine elastase I 1.6 X 10(10) M-1, and for Subtilisin Carlsberg 3.3 X 10(7) M-1. For isoinhibitors 2-5, the K alpha ranges were 7.1 X 10(10) to 1.3 X 10(11) M-1 for alpha-chymotrypsin, 1.0 X 10(9) to 5.6 X 10(9) M-1 for porcine elastase I, and 6.0 X 10(8) to 1.3 X 10(9) M-1 for subtilisin Carlsberg. Because of the strong affinity of these inhibitors for alpha-chymotrypsin and elastase, two proteins in the normal environment of the nematode, the name isoinhibitors of chymotrypsin/elastase is suggested for these proteins.

The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: The reactive site☆☆☆

Five isoinhibitors of chymotrypsin/elastase present in aqueous extracts of Ascaris were isolated. The reactive site in each isoinhibitor, the peptide bond that during encounter is positioned over the catalytic site in chymotrypsin, is Leu-Met. This bond was hydrolyzed by incubating intact isoinhibitors with 5-25 mol% chymotrypsin at pH 3.2 for 4-6 days (isoinhibitor 1) or 2.5-5 weeks (isoinhibitors 2-5). The reaction under these conditions did not proceed beyond 60% modified isoinhibitor (peptide bond hydrolyzed) and 40% intact inhibitor. The Leu-Met bond, hydrolyzed in modified isoinhibitor, can be resynthesized at pH 7.6 by incubating modified inhibitor with a stoichiometric amount of chymotrypsin bound to Sepharose CL-4B and then dissociating the complex in a kinetically controlled fashion with 5% trichloroacetic acid. The product, intact inhibitor, was obtained in greater than 80% yield. The site in the isoinhibitor that is positioned over the catalytic site in elastase during encounter is the same as for encounter with chymotrypsin. The Leu-Met bond hydrolyzed during encounter with elastase can be resynthesized by chymotrypsin. Chymotrypsin and elastase bind to the inhibitor at the same site.

PHYSIOLOGICAL CHARACTERIZATION OF TOXINS ISOLATED FROM SCORPION VENOM

ABSTRACT Chromatographie fractionation of venom from the scorpion, Centruroides sculpturatus , shows that this venom contains many low molecular weight proteins that have widely varying toxicities and biological actions. Thirteen of these proteins have been purified. Toxicities range from the highly lethal Toxins III and IV (LD 50 , chicks, 0.02 mg/kg) to the relatively non-lethal variants, variant 3 for example. A comparison of toxicities in chicks and crickets shows that these toxins are not necessarily host specific, e.g. Toxin III has an LD 50 o f 0.02 and 60 mg/kg in cockerels and crickets, respectively; Toxin IV an LD 50 of 0.02 and 1.86 mg/kg respectively; and Toxin 140–1 an LD 50 of 0.87 and 1.60 mg/kg in cockerels and crickets, respectively. The effects of each of the thirteen toxins upon neuromuscular transmission have been studied using the biventer cervicis muscle isolated from the chick. Three effects are observed: (1) block of the twitch upon indirect stimulation (e.g. toxin 144–4 at a dosage of I.85 μg/ml); (2) initial potentiation of the twitch followed by block (Toxin III, dosage 6.25 × 10 -3 μg/ml); and (3) initial potentiation with induction of an oscillation manifested in both the twitch and the resting tension (baseline tension) of the muscle (toxin 140–1, dosage 0.4 μg/ml). The oscillation consists of an accurately timed periodic contracture and relaxation (baseline oscillation) and, likewise, a periodic increase and decrease in the strength of the twitch upon indirect stimulation. A similar oscillation is shown by this muscle when the (Ca 2+ ) is less than 1.72 mM and without toxin. Venom is initially fractionated by column chromatography on CM-cellulose with ammonium acetate buffers of increasing pH and concentrations. The toxins are further purified by rechromatography, on other absorbents, of CMC eluates again using ammonium acetate buffers as developers. Amino acid sequences of four of these toxins have been reported (Babin et al., 1974;1975). Evidence from amino acid compositions suggest that possibly four or five of these isolates have only three disulfide bridges and 50–55 amino acid residues. The other isolates contain four disulfide bridges and 60-66 amino acid residues.

Structural studies on porcine hemopexin

1. Porcine hemopexin was isolated from the serum of a single animal and purified to homogeneity. 2. Porcine hemopexin has an apparent Mw of 67,000, binds heme in a 1:1 molar ratio and consists of 24% N-linked oligosaccharides. The amino acid composition of porcine hemopexin compares well with the amino acid composition of human and rabbit hemopexins. 3. Limited tryptic hydrolysis of apohemopexin generates stable peptides of apparent Mw 42,000, 25,000, 24,000 and 21,000. The tryptic peptide of apparent Mw 42,000 (peptide I) binds heme in a 1:1 molar ratio, consists of 33% N-linked oligosaccharides and is derived from the amino terminal of intact hemopexin. The three peptides of smaller-Mw (collectively peptide II) represent the carboxyl terminal half of hemopexin, do not contain N-linked oligosaccharides and have no heme-binding capability. The Mw heterogeneity of peptide II is likely due to cleavage at secondary sites. 4. Under nondissociating electrophoresis two bands are resolved for hemopexin and peptide I, indicating the possibility of polymorphism in porcine hemopexin.

Bovine serum hemopexin: properties of the protein from a single animal

1. Hemopexin was isolated from bovine serum of a single animal in a yield of 0.5 mg/ml. 2. Bovine hemopexin was found to exist in two isoforms of mol. wt 68,000 and 65,000. 3. Treatment of hemopexin with glycopeptidase F yields a single band corresponding to a mol. wt of 51,000. 4. The protein binds heme on an equimolar ratio and shows a single component in reverse-phase high performance liquid chromatography. 5. The amino acid composition of bovine hemopexin compares with that of hemopexin isolated form other animals.