Delineating residues for haemolytic activities of snake venom cardiotoxin 1 from Naja naja as probed by molecular dynamics simulations and in vitro validations.

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59-62 amino acids with four disulfide bridges and globular proteins of simple ß-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions ('head groove' and 'loop groove') of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana.

Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small ß-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of 13C,15N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

Cytolytic Activity and Molecular Feature of Cardiotoxin and Cardiotoxin-like Basic Protein: The Electrostatic Potential Field Is an Important Factor for Cell Lytic Activity.

BACKGROUND/AIM: Cardiotoxin (CT) is a well-known cell lytic protein and has been purified from cobra venom. Cardiotoxin-like basic protein (CLBP) has two amino acid insertions and does not exhibit cell lytic activity. The molecular features of these CT family proteins were examined in the present study using molecular modeling and molecular simulation techniques. MATERIALS AND METHODS: Molecular models of CT and CLBP were constructed based on the X-ray data of Naja mossambica mossambica CT VII4 (Protein Data Bank ID: 1CDT). The structural features of these models were examined using molecular orbital and electrostatic potential parameters. RESULTS: The stereo-hydrophobicities and molecular torsions of CT and CLBP, which are indexes of structural features, were similar. Electrostatic potential fields (ESP) differed between CT and CLBP and this was considered one of the critical factors in molecular titer. CONCLUSION: The distribution of ESP fields may affect the cytolytic activity of the CT family.

Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening.

Antiproliferative activity of cobra venom cytotoxins.

Cytotoxins (or cardiotoxins, CTs) are small rigid membrane-active proteins of the three-finger toxin (TFT) family. They comprise about 60 amino acid residues, stabilized by four disulphide bridges. CTs, the most abundant proteins in cobra venom are able to kill cancer cells in a dose and time-dependent manner. The present review summarizes the current data on the molecular pathways of cancer cell death, induced by CTs. A relationship between structural characteristics of CTs and mechanism of their antiproliferative activity is reviewed as well. The CT molecules are rigid and their structure does not change significantly, when they interact with their molecular targets. This rigidity facilitates identification of molecular entities, responsible for antiproliferative activity of the toxins. We demonstrate that consideration of a net electrostatic charge and recently introduced HTL (Hydrophobicity of the Tips of the Loops) score allows distinguishing between the two mechanisms of cell death. The first is related to membrane destabilization by the toxins. The second involves their capture inside the cells, followed by interrogation into signal cascades mediated by the proteins, essential for cell life. Via addressing to antibacterial activity of CTs, which is supposed to arise from the plasma membrane damage, we demonstrate that, if membrane deterioration is involved in malignant cell death, the toxic activity of CTs correlates with their HTL scores and net electrostatic charge. We assume the relationship found may be used for rational design of antiproliferative compounds.

Cardiotoxin III suppresses hepatocyte growth factor-stimulated migration and invasion of MDA-MB-231 cells.

The hepatocyte growth factor (HGF)/c-Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA-MB-231 cells. When cells were treated with non-toxic doses of CTX III, CTX III inhibited the HGF-promoted cell migration and invasion. CTX III significantly suppressed the HGF-induced c-Met phosphorylation and downstream activation of phosphatidylinositol 3-kinase (PI3k)/Akt and extracellular signal-regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF-κB) as well as a reduction of matrix metalloproteinase-9 (MMP-9) activity. Furthermore, the c-Met inhibitor PHA665752 inhibited HGF-induced MMP-9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c-Met activation. Taken together, these findings suggest that CTX III inhibits the HGF-induced invasion and migration of MDA-MB-231 cells via HGF/c-Met-dependent PI3K/Akt, ERK1/2 and NF-κB signalling pathways, leading to the downregulation of MMP-9 expression.

Inhibition of leukemic U937 cell growth by induction of apoptosis, cell cycle arrest and suppression of VEGF, MMP-2 and MMP-9 activities by cytotoxin protein NN-32 purified from Indian spectacled cobra (Naja naja) venom.

A cytotoxin NN-32 (6.7 kDa) from Indian cobra (Naja naja) venom inhibited human leukemic U937 cell growth as observed by Trypan blue dye exclusion method and cytotoxicity was confirmed by MTT assay. NN-32 induced apoptosis of U937 cell and cell cycle arrest of sub-G1 phase were revealed by FACS analysis. Increased Bax/Bcl-2 ratio, increased caspase 3 and 9 activities, cleaved PARP, decreased VEGF, MMP-2 and MMP-9 activities were observed after NN-32 treatment of U937 cell. Antileukemic activity of NN-32 on U937 cell may be due to activation of apoptosis, arresting cell cycle and antiangiogenesis activities.

Cardiotoxin-I: an unexpectedly potent insulinotropic agent.

Insulin secretion from pancreatic ß-cells is a complex process, involving the integration and interaction of multiple external and internal stimuli, in which glucose plays a major role. Understanding the physiology leading to insulin release is a crucial step toward the identification of new targets. In this study, we evaluated the presence of insulinotropic metabolites in Naja kaouthia snake venom. Only one fraction, identified as cardiotoxin-I (CTX-I) was able to induce insulin secretion from INS-1E cells without affecting cell viability and integrity, as assessed by MTT and LDH assays. Interestingly, CTX-I was also able to stimulate insulin secretion from INS-1E cells even in the absence of glucose. Although cardiotoxins have been characterized as potent hemolytic agents and vasoconstrictors, CTX-I was unable to induce direct hemolysis of human erythrocytes or to induce potent vasoconstriction. As such, this newly identified insulin-releasing toxin will surely enrich the pool of existing tools to study ß-cell physiology or even open a new therapeutic avenue.

Improved method for the isolation, characterization and examination of neuromuscular and toxic properties of selected polypeptide fractions from the crude venom of the Taiwan cobra Naja naja atra.

An improved chromatographic method was developed to isolate and purify polypeptides and proteins from the crude venom of the Taiwan cobra Naja naja atra. The procedure devised is simple, easy to reproduce, and enables large scale isolation of almost all polypeptides and proteins in this cobra venom. Six pure polypeptide fractions of the venom were isolated and characterized using gel filtration on Sephadex G50 (medium), ion exchange chromatography on SP-Sephadex C25, desalting on Sephadex G25 (fine) and preparative HPLC on a RPC 18 column. The neuromuscular activity of these fractions was tested on the chick biventer cervicis nerve-muscle preparation and their toxicity (LD(50)) was determined after i.v. administration in mice. Their antinociceptive activity was tested in the mouse abdominal test by i.v. application. Two of these polypeptide samples had major physiological effects: one acted as a cardiotoxin causing reversible myocardial contractures with no effect on muscle twitches elicited by nerve stimulation (NS); another was a neurotoxin that blocked muscle contractions in response to NS and exogenously added acetylcholine. The cardiotoxic fraction was identified as CTX I, a well-known cardiotoxin present in this venom, and the neurotoxin was identified as neurotoxin-α with an LD50 in mice of 0.075 mg/kg.

A lethal cardiotoxic-cytotoxic protein from the Indian monocellate cobra (Naja kaouthia) venom.

A lethal cardiotoxic-cytotoxic protein (mol. wt. 6.76 kDa) has been purified from the Indian monocellate cobra (Naja kaouthia) venom by ion-exchange chromatography and HPLC. CD spectra indicated the presence of 23% alpha helix, 19% beta sheets and 35% coil. Complete amino acid sequence was determined by MALDI, which showed similar homology with cardiotoxins/cytotoxins isolated from venom of other Naja species. Intraperitoneal LD(50) was 2.5 mg kg(-1) in BalbC male mice. In vitro cardiotoxicity studies on isolated guinea pig auricle showed that the molecule produced auricular blockade that was abolished after trypsin treatment. Cytotoxicity studies on human leukemic U937 and K562 cells showed that it significantly inhibited cell proliferation in a dose and time dependent manner, as observed by trypan blue exclusion method and tetrazolium bromide reduction assay. IC(5)(0) on U937 and K562 cells were 3.5 microg/ml and 1.1 microg/ml respectively. Morphometry and cell sorting studies indicated apoptosis induction in toxin treated leukemic cells. Apoptosis was caspase 3 and 9 dependent and the treated leukemic cells were arrested in sub-G1 stage. There was an increase in Bax-Bcl2 ratio, decrease in HSP (Heat shock protein) 70 and HSP90 and induction of PARP cleavage after NK-CT1 treatment. The toxin showed low cytotoxic effect on normal human leukocytes as compared with imatinib mesylate. Further detailed cytotoxic and cardiotoxic effects at the molecular level are in progress.

Taiwan cobra cardiotoxin III inhibits Src kinase leading to apoptosis and cell cycle arrest of oral squamous cell carcinoma Ca9-22 cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60-amino acid residues isolated from Naja naja atra venom, has been reported to have cytotoxic activity. CTX III exerted cytotoxicity with the S-phase cell cycle arrest, correlated with a marked decrease in the expression levels of cyclin A, cyclin B, and cyclin-dependent kinase 1 (CDK1), and apoptosis, accompanied with Bax and Bad up-regulation, and the down-regulation of Bcl-2, p-Bad, and X-linked inhibitor of apoptosis (XIAP) with cytochrome c release and sequential activation of caspase-9 and caspase-3 in Ca9-22 cells. Mechanistic studies showed that CTX III suppressed the phosphorylation of Src, EGFR, STAT3, STAT5, Akt, and activation of PI3 K (p110). Moreover, Src inactivation was observed earlier than that of the EGFR and the Src inhibitor PP2 suppressed the levels of phospho-EGFR, phospho-STAT3, phospho-STAT5, phospho-Akt, and PI3 K(p110). The PP2 also caused the S-phase arrest and apoptosis, and led to down-regulation of Bcl-2, p-Bad, XIAP, cyclin A, cyclin B, and CDK1, and up-regulation of Bax and Bad, similar to that observed in CTX III treatment. Taken together, these results indicate that CTX III induces apoptosis and S-phase arrest in Ca9-22 cells via concomitant inactivation of the Src, EGFR, STAT3, STAT5, PI3 K(p110), and Akt signaling pathways.

Effects of cardiotoxin III on NF-kappaB function, proliferation, and apoptosis in human breast MCF-7 cancer cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced apoptosis in human breast MCF-7 cancer cells was confirmed by sub-G1 formation, phosphatidylserine (PS) externalization, and poly (ADP-ribose) polymerase (PARP) cleavage with an IC50 of 2 microg/ml at 48 h. Effects of CTX III on proliferation and apoptosis correlated with upregulation of Bax, and downregulation of Bcl-XL, Bcl-2, and XIAP, with no appreciable alteration on the protein levels of Bid, Bim, and survivin. CTX III treatment also caused release of mitochondrial cytochrome c to the cytosol, which led to subsequent activation of capase-9. Moreover, CTX III inhibited the nuclear factor-kappaB (NF-kappaB) activation through inhibition of IkappaB kinase (IkappaK) activity. Overall, our results indicate that CTX III downregulates NF-kappaB in MCF-7 cells, leading to the suppression of proliferation and induction of apoptosis. These findings suggest the molecular basis for CTX III-induced apoptotic death of MCF-7 cells.

Apoptosis of human hepatocellular carcinoma cell (HepG2) induced by cardiotoxin III through S-phase arrest.

Cytotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, have potential therapeutic activity in tumor therapy. However, the therapeutic effect in solid tumor treatment with CTX III are still largely unknown. In the present study, we investigated whether CTX III affects cell growth and cell cycle progression of hepatocellular carcinoma cell (HepG2). We found that the proliferation of HepG2 cell was inhibited by CTX III, to some extent, in a time- and dose-dependent manner (IC50 2.58microg/ml at 24h). Flow cytometric analysis and annexin V labeling also demonstrated that CTX III increased the percentage of apoptotic cells being associated with cell cycle arrest at S-phase. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot revealed that cyclin D1, cyclin A and cyclin E, which involved in cell apopotosis and cell cycle progression, were down regulated both at transcription and translation levels. CTX III-induced caspase-8, -9 and caspase-3 activation, generation of truncated Bid, releasing of cytochrome c and the change of Bcl-2/Bax ratio on protein and mRNA levels. These findings demonstrated that cyclin D1, cyclin B and cyclin A down-regulation, change of Bcl-2/Bax ratio and caspase-8 and -9 activation contribute to CTX III-induced HepG2 cell apoptosis.

Structures of heparin-derived tetrasaccharide bound to cobra cardiotoxins: heparin binding at a single protein site with diverse side chain interactions.

Cobra cardiotoxins (CTXs) are three-fingered polypeptides with positively charged domains that have been shown to bind to anionic ligands of snake venom citrate, glycosaminoglycans, sulfoglycosphingolipid, and nucleotide triphosphate with various biochemical effects including toxin dimerization, cell surface retention, membrane pore formation, cell internalization and blocking of enzymatic activities of kinase and ATPase. The reported anionic binding sites, however, are found to be different among different CTX homologues for potentially different CTX activities. Herein, by NMR studies of the binding of inorganic phosphate, dATP (stable form of ATP), and heparin-derived tetrasaccharide to Naja atra CTX A1, a novel CTX molecule exhibiting in vivo necrotic activity on skeletal muscle, we demonstrate that diverse ligands binding to CTXs could also occur at a single protein site with flexible side chain interactions. The flexibility of such an interaction is also illustrated by the available heparin-CTX A3 complex structures with different heparin chain lengths binding at the same site. Our results provide a likely structural explanation on how the interaction between heparan sufate and proteins depends more on the overall charge cluster organization rather than on their fine structures. We also suggest that the ligand binding site of CTX homologues can be fine-tuned by nonconserved residues near the binding pocket because of their flexible side chain interaction and dimerization ability, even for the rigid CTX molecules tightened by four disulfide bonds.

Involvement of c-jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by cardiotoxin III (Naja naja atra) in K562 leukemia cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, may have a potentiality as a structural template for rational drug design in killing cancer cells. Treatment of K562 cells with 0.3 microM of CTX III resulted in G2/M phase cell cycle arrest that was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin A, cyclin B1, Cdk2 and Cdc25C. In contrast to no effect on the phosphorylation of ERK, p38 MAPK and Akt, an activation of JNK was noted when K562 cells were exposed to CTX III. CTX III-mediated G2/M phase arrest and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125, but not by ERK and p38MAPK inhibitors. Further investigation showed that the specific JNK inhibitor, SP600125, reduced the activation of caspase-3, caspase-9, and reversed the decline in the expression of cyclin B1. Taken together, our data show for the first time that JNK, but not ERK, p38MAPK or Akt signaling, plays an important role in CTX III-mediated G2/M arrest and apoptosis in K562 cancer cells.

Purification, partial characterization, crystallization and preliminary X-ray diffraction of a novel cardiotoxin-like basic protein from Naja naja atra (South Anhui) venom.

A novel cardiotoxin-like basic protein was isolated from the venom of the Chinese cobra (Naja naja atra) from the south of Anhui in China. The protein inhibits the expression of vascular endothelial growth factor and basic fibroblast growth factor in human lung cancer cell line H1299 and induces the haemolysis of rabbit erythrocytes under low-lecithin conditions. After a two-step chromatographic purification, the resultant 7 kDa protein was crystallized by the hanging-drop vapour-diffusion method at room temperature. A complete data set was collected to 2.35 A resolution using an in-house X-ray diffraction system. The crystal belongs to space group P4(1)2(1)2, with unit-cell parameters a = b = 43.2, c = 147.9 A. There are two molecules in the crystallographic asymmetric unit.

Purification and characterization of Ophiophagus hannah cytotoxin-like proteins.

Three cytotoxin-like proteins from the venom of Ophiophagus hannah were isolated by a combination of ion exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that these proteins all consisted of 63 amino acids and shared approximate 50% and 56% sequence identity with Naja naja atra cardiotoxins and cardiotoxin-like basic proteins (CLBPs), respectively. CD spectra revealed that their secondary structure was dominated with beta-sheet as those noted with cardiotoxins and CLBPs. O. hannah cytotoxin-like protein exhibited a cell-lytic activity on SK-N-SH cells, but its activity was more weak than that noted for N. naja atra cardiotoxin 3. Alternatively, apoptotic cell death was induced by the addition of N. naja atra CLBP. Based on the sequence information with the toxin molecules, the functional residues and regions related to the differential activity with O. hannah cytotoxin-like protein, cardiotoxin and CLBP are discussed.

Occurrence of non-protein low molecular weight cardiotoxin in Indian King Cobra (Ophiophagus hannah) Cantor 1836, venom.

Pathophysiology due to snakebite is a combined effect of various actions of the complex venom constituents. Importance of protein toxins in snake envenomation is well known. The present investigation reports the existence of nonprotein/nonpetide low molecular weight toxin in Indian King Cobra venom, which plays an important role in envenomation consequences in experimental animal models. A group of non-peptidic toxins (OH-NPT1) was isolated from Indian King Cobra Ophiophagus hannah by thin layer chromatography and silica gel column chromatography. UV, IR, NMR and (ESI) TOF-MS studies characterized the OH-NPT1 as a mixture of aliphatic acids having molecular weights 256, 326 and 340Da. The minimum lethal dose of OH-NPT1 was found to be 2.5 microg/20g (iv) and 4microg/20g (ip) in male albino mice. The cardiotoxic property of OH-NPT1 was established through studies on isolated guinea pig heart and auricle preparations, ECG studies in albino rat and estimation of LDH1/LDH and CPK-MB/CPK ratio in Swiss albino mice. Commercial antiserum failed to neutralize the lethality and cardiotoxicity of the toxin. However, calcium and magnesium effectively neutralized the lethal action.

Structural difference between group I and group II cobra cardiotoxins: X-ray, NMR, and CD analysis of the effect of cis-proline conformation on three-fingered toxins.

Natural homologues of cobra cardiotoxins (CTXs) were classified into two structural subclasses of group I and II based on the amino acid sequence and circular dichroism analysis, but the exact differences in their three-dimensional structures and biological significance remain elusive. We show by circular dichroism, NMR spectroscopic, and X-ray crystallographic analyses of a newly purified group I CTX A6 from eastern Taiwan cobra (Naja atra) venoms that its loop I conformation adopts a type VIa turn with a cis peptide bond located between two proline residues of PPxY. A similar "banana-twisted" conformation can be observed in other group I CTXs and also in cyclolinopeptide A and its analogues. By binding to the membrane environment, group I CTX undergoes a conformational change to adopt a more extended hydrophobic domain with beta-sheet twisting closer to the one adopted by group II CTX. This result resolves a discrepancy in the CTX structural difference reported previously between solution as well as crystal state and shows that, in addition to the hydrophobicity, the exact loop I conformation also plays an important role in CTX-membrane interaction. Potential protein targets of group I CTXs after cell internalization are also discussed on the basis of the determined loop I conformation.

Immobilized betulinic acid column and its interactions with phospholipase A2 and snake venom proteins.

Betulinic acid (BA) is a plant-derived pentacyclic triterpenoid. Although BA has been found to have diverse pharmacological effects, including anti-tumor and anti-inflammatory actions and potential as inhibitor of phospholipase A2 (PLA2), its cellular targets remain unclear. In this study, BA was immobilized onto an acrylamide matrix. The immobilized-BA column could retain the purified PLA2 of bovine pancreas or the PLA2 of snake venom from Naja nigricollis. The bound PLA2 were not eluted by high salt concentrations but were eluted by either acid or calcium free buffer. Besides the PLA2, a group of basic proteins of snake venom with molecular weights of about 7 kDa were also strongly bound by immobilized BA. One of these proteins was identified as gamma-cardiotoxin. The usefulness of immobilized BA for exploring the cellular targets of BA is discussed.