Subunit Of Crotoxin Research Articles

Effects of chemical modifications of crotoxin B, the phospholipase A 2 subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities

Crotoxin B, the basic Asp49-PLA 2 subunit from crotoxin, the main component of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagulant activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitrophenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inhibition with ethylenediaminetetraacetic acid (EDTA), were carried out in order to study their effects on enzymatic and pharmacological activities. Lethality was reduced after modification of His or Lys residues, as well as after cleavage with CNBr, while enzymatic activity was completely abolished after modification of His or incubation with EDTA. Modification of Lys or Tyr, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagulant activity was modified similarly to enzymatic activity, evidencing the dependency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage with CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal effect was significantly reduced only after modification of Lys and after cleavage with CNBr. Edema-inducing activity was partially inhibited after treatment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced after modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from the active catalytic site which are involved in some of the toxic and pharmacological effects induced by this phospholipase A 2.

Isolation and characterization of novel neurotoxins from south american rattlesnake crotalus durissus terrificus: Optimization of chromatographic procedure

Abstract: Molecular exclusion HPLC chromatography on a Protein Pak SW 300 column and reversed phase HPLC on a µ-Bondapack Cl8 column were used to purify four novel neurotoxins (rom the crotoxin complex, and five crotoxin subunits (PLA2 and crotapotin) as well as two crotamine isoforms. A single preparative reversed phase HPLC steps yielded a major basic myotoxin followed by the subunits of crotoxin (three PLA2 isoforms and two crotapotin isoforms). This modified procedure may be useful for the rapid, large-scale purification of toxins from Crotalid venoms.

Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (Maracambóia) venom.

The PLA2 and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of high-performance liquid chromatography (HPLC) molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2 and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2 showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2 showed allosteric behavior, with maximal activity at pH 8.3 and 35-40 degrees C. C. d. cascavella PLA2 required Ca2+ for activity but was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2 by acting as allosteric inhibitors.

Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (Maracambóia) venom.

The PLA2 and crotapotin subunits of crotoxin from Crotalus durissus cascavella venom were purified by a combination of HPLC molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2 and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2 showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2 showed allosteric behavior, with maximal activity at pH 8.3 and 35-40 degrees C. The C. d. cascavella PLA2 required Ca2+ for activity, but was inhibited by Cu2+ and Zn2+ and by Cu2+ and Mg2+ in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2 by acting as allosteric inhibitors.

Molecular mimicry between a monoclonal antibody and one subunit of crotoxin, a heterodimeric phospholipase A2 neurotoxin.

Crotoxin is a heterodimeric phospholipase A2 neurotoxin formed by the non-covalent association of an acidic and non-toxic subunit, CA, and a basic and weakly toxic phospholipase A2, CB. The two subunits behave in a synergistic manner. CA enhances the lethal potency of CB by increasing its selectivity of action. The mAb A-56.36, directed against the non-toxic subunit CA, was previously shown to neutralize crotoxin toxicity by dissociating the crotoxin complex. In the present report, a polypeptide sequence similarity was observed between some CDRs of mAb A-56.36 and two regions of CB (pos. 60-80 and 95-110). Phage displayed peptides corresponding to VH2 and VH3 of mAb A-56.36 and to their homologous sequences in CB bind CA to different extents. This observation shows that mAb A-56.36 interacts with a region of CA involved in its interaction with CB, therefore mimicking the binding of CB to CA. A similar approach was used to determine the regions of ammodytoxin A and of agkistrodotoxin, two phospholipase A2 neurotoxins similar to CB, which are involved in the formation of heterocomplexes with CA. The analysis of these data contributes to the determination of stretches of amino acids which could constitute the paratope of mAb A-56.36, as well as the region of association of CB with CA in crotoxin.

Effect of Crotalus venom on the humoral and cellular immune response

The effect of venom of South American rattlesnake Crotalus durissus terrificus (cdt) on the humoral and cellular immune response was studied in BALB/c mice that were immunized with soluble antigens [human serum albumin (HSA) or chicken ovoalbumin (OVA)]or sensitized to DNFB 1 hr after venom injection. Pretreatment of the animals with cdt venom induced a significant reduction in the level of anti-OVA and anti-HSA IgG antibodies. The effect of crotoxin, a major neurotoxic component of cdt venom, its acidic non-toxic subunit (CA) and its basic phospholipase A 2 subunit (CB) was also studied. The whole crotoxin molecule was as able as cdt venom to induce a significant decrease in the level of anti-OVA and anti-HSA IgG antibodies. However, the CA and CB subunits of crotoxin did not change the antibody level to either antigen, suggesting that the suppressive effect of crotoxin requires the intact molecule. Both cdt venom and the whole crotoxin molecule were able to induce a significant decrease in the level of anti-HSA IgG1 antibodies. The levels of other IgG isotypes and IgE were barely detectable and could not be estimated. In spite of their suppressive effect on the humoral immune response neither cdt venom nor crotoxin had any effect on the cellular immune response as estimated by contact sensitivity reaction to DNFB. It is suggested that cdt venom and its crotoxin component have an inhibitory effect on the humoral but not on the cellular immune response.

Re-examination of crotoxin-membrane interactions

The interaction of crotoxin with synaptic membranes from Torpedo marmorata has been re-examined, using radioiodinated toxin. In competition experiments, the ‘saturable binding’ is usually calculated by subtracting the non-saturable binding, determined in the presence of an excess of unlabelled crotoxin, from total binding. Paradoxically, we observed a notable increase of the ‘saturable binding’ of 125I-crotoxin, defined in this manner in the presence of a high concentration of certain competitors (e.g. crotoxin subunit B, agkistrodotoxin, ammodytoxin and ammodytin I 2). This potentiation effect was analysed by competition and cross-linking experiments. The dissociation of the basic crotoxin subunit CB, which differs markedly from crotoxin in its membrane- binding characteristics, was found to be the main reason for the observed phenomenon. 125I-CB could be released from 125I-crotoxin by exchange with a molecule having sufficient affinity towards CA (e.g. CB or agkistrodotoxin) or it could be dissociated from the specific crotoxin membrane-binding site(s) by a competitor molecule. Our results, therefore, suggest a reinterpretation of previous 125I-crotoxin binding studies.

Double role of the chaperon subunit of crotoxin

Double role of the chaperon subunit of crotoxin

Binding proteins on synaptic membranes for crotoxin and taipoxin, two phospholipases A 2 with neurotoxicity

Crotoxin and taipoxin are both neurotoxic phospholipases A 2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A 2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A 2. Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A 2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca 2+-free condition, taipoxin or crotoxin inhibits with ic 50 of 20–1000 nM the Na +-dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected. Furthermore, the high-affinity site for [ 3H]desipramine binding, known to be the norepinephrine transporter, is inhibited with an ic 50 of 14nM by taipoxin independent of phospholipase activity. These results are strong indications that certain synaptosomal biogenic amine transporters are part of the binding proteins for taipoxin and crotoxin. Chemical modification at Tyr-21 of the phospholipase subunit of crotoxin greatly reduces the neurotoxicity and the binding affinity with little effect on the enzymatic activity of the toxin. Hence, Tyr-21 may be an important residue for the binding of crotoxin and perhaps other phospholipase A 2 neurotoxins. Even stronger evidence comes from the finding that replacement of the corresponding Phe residue of bovine pancreatic phospholipase A 2 with Tyr by site-directed mutagenesis enables it to compete with an ic 50 of 1 μM for the binding of [ 125I]crotoxin, contrasting sharply with the complete lack of such ability with the wild type even at 50 μM. In addition, rat phospholipase A 2, which has a corresponding Tyr residue, can inhibit the binding of [ 125I]crotoxin.

Double role of the non-toxic and non-enzymatic subunit of crotoxin in the toxin mechanism of action

Double role of the non-toxic and non-enzymatic subunit of crotoxin in the toxin mechanism of action

Antipeptide antibodies directed to the C-terminal part of ammodytoxin A react with the PLA 2 subunit of crotoxin and neutralize its pharmacological activity

V. C̆urin-S̆erbec, E. Délot, G. Faure, B. Saliou, F. Gubenšek, C. Bon and V. Choumet. Antipeptide antibodies directed to the C-terminal part of ammodytoxin A react with the PLA 2 subunit of crotoxin and neutralize its pharmacological activity. Toxicon 32, 1337–1348, 1994.—Crotoxin and ammodytoxin A are snake venom neurotoxic phospholipases A 2. Polyclonal antibodies against three synthetic peptides selected from the C-terminal part of the primary structure of ammodytoxin A were tested by ELISA for their interaction with crotoxin and its subunits, CA and CB. All three antipeptide antibodies reacted specifically with corresponding parts of ammodytoxin A and CB, either native or reduced. Conversely, polyclonal antibodies produced against ammodytoxin A and CB reacted strongly with all three peptides, suggesting that they constitute at least a part of natural epitopes in both proteins. All antipeptide antibodies reacted also with the corresponding peptides derived from CB by cyanogen bromide cleavage. The biological activity of the immune complexes was tested. No significant change in the enzymatic activity of CB, ammodytoxin A or crotoxin was observed with any of the three antipeptide antibodies. These antibodies were, however, able to protect mice against the lethal potency of CB and to prolong survival time of mice injected with crotoxin. These antipeptide antibodies were assayed in vitro for their protective effect against the action of CB or crotoxin on synaptosomes from Torpedo marmorata electric organ. They partly inhibited the acetylcholine release induced by both proteins. These results indicate that the C-terminal part of CB is likely to be involved in the pharmacological action of crotoxin.

The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus

Crotoxin, the main toxin from the venom of the South American rattlesnake Crotalus durissus terrificus, is a beta-neurotoxin which consists of the non-covalent association of two subunits: a phospholipase A2 subunit B (CB), and a non-enzymic subunit A (CA). We have previously purified and characterized several isoforms of each subunit of crotoxin in the venom collected from numerous snakes. Furthermore, three cDNAs encoding two CB isoforms and the precursor, pro-CA, of subunit A have been isolated from a cDNA library prepared from a single venom gland of Crotalus durissus terrificus. The aim of this study is to analyse an individual snake venom from an animal that has been used to construct a cDNA library. Several isoforms of subunit A and two isoforms of subunit B were isolated and compared to purified and characterized subunit isoforms from pooled venom. The result of this study showed that the multiplicity and the diversity of crotoxin isoforms result from post-translational modifications occurring on a precursor and from the expression of different messenger RNAs present in an individual snake. It allowed for the identification of the two CB isoforms encoding cDNAs expressed in the individual venom with two isoforms from pooled venom, CBc and probably CBa2, that belong to two classes of crotoxin complexes which can be distinguished biochemically and pharmacologically.

Model for the interaction of crotoxin, a phospholipase A2 neurotoxin, with presynaptic membranes.

Crotoxin is a phospholipase A2 neurotoxin that impairs the release of acetylcholine at neuromuscular junctions, primarily at the presynaptic level. It associates a phospholipase A2 subunit, CB, with a chaperon subunit, CA. We have shown elsewhere that the purely cholinergic synaptosomes from the Torpedo electric organ provided a convenient model to study the pharmacology of crotoxin and other related neurotoxins [Délot, E., & Bon, C. (1992) J. Neurochem. 58, 311-319]. In the present experiments, we labeled crotoxin with 125I and demonstrated saturable binding to Torpedo presynaptic membranes. In the range of concentrations that was effective on synaptosomes, crotoxin bound to a single class of sites without cooperativity. The binding was affected by divalent cations, and its kinetics was rather complex. We observed a competition between crotoxin and related neurotoxins, but not CB. Although CA could not bind, it could compete efficiently with crotoxin. We compare our results with those previously obtained by others on guinea pig brain membranes. On Torpedo membranes, as on all models tested, CB was the major species bound to the membrane, while CA remained in solution. However, the mechanism underlying this phenomenon had never been clarified. The major question is the time scale of the events, i.e., does CB bind before or after dissociating from CA? Our results indicate that the predominant pathway involves the formation of a ternary complex between crotoxin's subunits and the acceptor site preceding the release of CA.

Determination of solution structure of CA3, subunit of crotoxin: A. Mikou, 1Y. Yang, 1A. Thomas, 1E. Guittet, 1J. Y. Lallemand, 1G. Faure and C. Bon ( 1Laboratoire de RMN, ICSN-CNRS, Gif-sur-Yvette, France; and 2Unité des Venins, Institut Pasteur, Paris, France)

Determination of solution structure of CA3, subunit of crotoxin: A. Mikou, 1Y. Yang, 1A. Thomas, 1E. Guittet, 1J. Y. Lallemand, 1G. Faure and C. Bon ( 1Laboratoire de RMN, ICSN-CNRS, Gif-sur-Yvette, France; and 2Unité des Venins, Institut Pasteur, Paris, France)

Cytotoxicity of crotoxin on murine erythroleukemia cellsin vitro

The cytotoxic effect of crotoxin, a heterodimeric phospholipase A2 from the venom of Crotalus durissus terrificus, was examined on murine erythroleukemia cells in vitro. Crotoxin cytocidal effect on cell growth had an EC50 of approximately 0.1-0.2 microM (3.0-5.0 micrograms/ml) in serum-free medium. Cytotoxicity was independent of cell growth since both quiescent and proliferating cells had similar sensitivities to the toxin. Dissociation of the crotoxin complex and phospholipase A2 activity of its subunit B are required for cytotoxicity, since the covalently linked crotoxin complex or the specific alkylation of the active site on the subunit B abolish the cytotoxic activity on murine erythroleukemia cells. Specific interaction between crotoxin and murine erythroleukemia cells appears to be required since the homologous phospholipase A2 from Crotalus atrox venom, with a higher phospholipase A2 specific activity than crotoxin, was 86-fold less potent than crotoxin. The data in this report show that the cytotoxic effect of crotoxin on murine erythroleukemia cells is consistent with the specific binding of the toxin resulting in cytocidal action mediated by the phospholipase A2 activity of crotoxin subunit B.

Snake-venom phospholipase A2 neurotoxins. Potentiation of a single-chain neurotoxin by the chaperon subunit of a two-component neurotoxin.

The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurotoxins (beta-neurotoxins): single-chain beta-neurotoxins, such as agkistrodotoxin and ammodytoxin-A, and dimeric beta-neurotoxins, which, in the case of the best studied ones, crotoxin-like toxins, consist of the non-covalent association of a phospholipase A2 (CB) and a non-enzymatic chaperon (CA). Possible evolutionary relationships of these beta-neurotoxins have been investigated by analyzing whether CA could behave as a chaperon toward agkistrodotoxin and ammodytoxin, as it does in the crotoxin complex. CA increased the lethal potency of agkistrodotoxin and modified its pharmacological effect on Torpedo synaptosomes. Sedimentation experiments proved that CA can form an heterocomplex with agkistrodotoxin. Agkistrodotoxin prevented the binding to CA of an anti-CA mAb which recognizes an epitope at the zone of interaction between crotoxin subunits, suggesting the association of CA and agkistrodotoxin implicated the same zone. A 10-fold molar excess of CA over ammodytoxin modified the effect of ammodytoxin on acetylcholine release but did not increase the lethal potency of ammodytoxin. Sedimentation experiments showed CA and ammodytoxin can form an heterocomplex which is less stable than CA.agkistrodotoxin. Ammodytoxin A did not compete with the anti-CA mAb. These observations are in good agreement with the sequence similarities between CB and agkistrodotoxin (80%) and ammodytoxin A (60%).

Immunochemical analysis of a snake venom phospholipase A 2 neurotoxin, crotoxin, with monoclonal antibodies

Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus. The crotoxin molecule is composed of two subunits: a basic and weakly toxic phospholipase A 2 (PLA 2) called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit called component-A (CA). Crotoxin exists as a mixture of several isoforms (or variants) resulting from the association of several subunit isoforms. We prepared monoclonal antibodies (MAbs) against each isolated subunit. Six anti-CA MAbs and eight anti-CB MAbs were tested for their cross-reactivities with each subunit and with other toxic and nontoxic PLA 2s. Four of the six anti-CA MAbs cross-reacted with CB, whereas only one of the eight anti-CB MAbs cross-reacted with CA. Two anti-CB MAbs were found to cross-react with agkistrodotoxin, a single chain neurotoxic PLA 2 purified from the venom of Agkistrodon blomhoffii brevicaudus. We determined the dissociation constants of each MAb for CA and CB isoforms and their capacities to neutralize the lethality and to inhibit the catalytic activity of crotoxin. We defined three epitopic regions on CA and four on CB, and used a schematic representation of the two subunits to characterize these epitopic regions with respect to: (1) the “toxic” and the “catalytic” sites of CB, and (2) the zone of interaction between the two subunits. We propose three-dimensional structures of the crotoxin subunits in which we localize amino acid residues that might be involved in the epitopic regions described here.

Analysis of cDNAs encoding the two subunits of crotoxin, a phospholipase A 2 neurotoxin from rattlesnake venom: the acidic non enzymatic subunit derives from a phospholipase A 2-like precursor

We report the sequences of three cDNAs encoding the two subunits (CA and CB) of crotoxin, a neurotoxic phospholipase A 2 from the venom of the South-American rattlesnake Crotalus durissus terrificus. CB is a basic and toxic phospholipaseA 2 and CA is an acidic, non toxic and non enzymatic three chain containing protein which enhances the lethal potency of CB. Two cDNAs encoding precursors of CB isoforms have been isolated from a cDNA library prepared from one venom gland. Both precursors are made of the same 16 residues signal peptide followed by a polypeptide of 122 amino acid residues. The two mature sequences differ from each other at eight positions and are in good agreement with the previous polypeptide sequence reported for CB. In the case of CA, the cDNA encodes a signal peptide identical to those found in CB precursors, followed by a polypeptide of 122 amino acids clearly homologous to phospholipases A 2 and including three regions which correspond to the three chains of mature CA. This demonstrates that CA is generated from a phospholipase A 2-like precursor, called pro-CA, by the removal of three peptides, leaving unchanged the molecule core cross-linked by disulfide bridges. The 5′-unttranslated tracts of cDNAs encoding CA and CB are nearly identical and the 3′-untranslated tracts are very similar, suggesting that the mRNAs encoding the two crotoxin subunits may result from the alternative splicing of a single gene or from the existence of a recent gene conversion. Data have been analysed in light of recent results on other phospholipase A 2 from different origins.

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes. Toxicon 29, 973–988 1991.—Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guineapig ( Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, ( K d1 = 4 nM and K d2 = 87 nM, B max1 = 2 and B max2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau ( K d = 2 nM and B max = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a K d-value of 3 nM. The K d value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, β-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A 2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A 2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.

The amino acid sequence of the acidic subunit B-chain of crotoxin

The B-chain of the acidic subunit of crotoxin proved refractory to Edman degradation. When subjected to sequence analysis using tandem mass spectrometry, pyroglutamate was found at the amino-terminal end, even though earlier attempts to de-block with pyroglutamate aminopeptidase were unsuccessful. The B-chain contained 35 amino acids and showed 91% amino acid identity with the corresponding segment from Mojave toxin, a homologous neurotoxin from Crotalus scutulatus scutulatus. The sequence of the last 24 residues of the B-chain is consistent with that previously published (Aird, S.D., Kaiser, I.I., Lewis, R.V. and Kruggel, W.G. (1985) Biochemistry 24, 7054–7058), except at position 20, where Edman degradation gave glycine and mass spectrometry gave glutamic acid.