Trimeresurus purpureomaculatus venom acetylcholinesterase has been partially purified by Sephadex G-200 gel filtration chromatography and DEAE Sephacel ion exchange chromatography. The enzyme has a mol. wt of 58,600. It was strongly inhibited by physostigmine salicylate and edrophonium chloride and exhibited substrate inhibition at high substrate concentration. The content of acetylcholinesterase in Trimeresurus purpureomaculatus venom was estimated to be much less than 0.3%.
The enzymatic activities of four samples of Malayan cobra venom were investigated. There was significant variation in the contents of L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, phospholipase A, 5'-nucleotidase and hyaluronidase. The phosphodiesterase content was, however, constant. Storage of the lyophilized venom powder at 25 degrees C for 1 month did not affect the enzymatic activities. The venom enzymatic activities were generally also stable at 4 degrees C in 0.85% saline solution. After incubation at 37 degrees C for 39 days in 0.85% saline solution, the venom still retained considerable amounts of enzymatic activities. SP-Sephadex C-25 ion-exchange chromatography of the venom showed that the phospholipase A, L-amino acid oxidase, 5'-nucleotidase, phosphodiesterase and alkaline phosphomonoesterase exist in multiple forms.
Tropidolaemus wagleri and Cryptelytrops purpureomaculatus are venomous pit viper species commonly found in Malaysia. Tandem mass spectrometry analysis of the crude venoms has detected different proteins in T. wagleri and C. purpureomaculatus. They were classified into 13 venom protein families consisting of enzymatic and nonenzymatic proteins. Enzymatic families detected in T. wagleri and C. purpureomaculatus venom were snake venom metalloproteinase, phospholipase A₂, ʟ-amino acid oxidase, serine proteases, 5'-nucleotidase, phosphodiesterase, and phospholipase B. In addition, glutaminyl cyclotransferase was detected in C. purpureomaculatus. C-type lectin-like proteins were common nonenzymatic components in both species. Waglerin was present and unique to T. wagleri-it was not in C. purpureomaculatus venom. In contrast, cysteine-rich secretory protein, bradykinin-potentiating peptide, and C-type natriuretic peptide were present in C. purpureomaculatus venom. Composition of the venom proteome of T. wagleri and C. purpureomaculatus provides useful information to guide production of effective antivenom and identification of proteins with potential therapeutic applications.
1. The biological properties of venoms from juvenile and adult common tiger snakes (Notechis scutatus) were compared. 2. The lethality, procoagulant activity and enzymatic activities of the juvenile venom were not substantially different from those of the adult venom. 3. Electrophoretic studies, however, indicated some minor differences in the protein composition of the juvenile and adult venoms.
The L-amino acid oxidase (EC 1. 4. 3. 2) from King cobra (Ophiophagus hannah) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was determined to be 140000 when examined by gel filtration and 68000 by SDS-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point of 4.5 and an intravenous LD50 of 5 micrograms/g in mice. It is a glycoprotein and contains two moles of FAD per mole of enzyme. The enzyme exhibited unusual thermal stability and unlike most other venom L-amino acid oxidases, it was stable in alkaline solution and was not inactivated by freezing.
Bungarus candidus venom exhibited high hyaluronidase, acetylcholinesterase and phospholipase A activities; low proteinase, 5'-nucleotidase, alkaline phosphomonoesterase and phosphodiesterase activities and moderately high L-amino acid oxidase activity. SP-Sephadex C-50 ion exchange chromatographic fractionation of the venom and Sephadex G-50 chromatography of the major lethal venom fractions indicate that the venom contains at least two highly lethal, basic phospholipases A with LD50 (i.v.) values of 0.02 micrograms/g (F6A) and 0.18 micrograms/g (F4A), respectively; as well as two polypeptide toxins with LD50 (i.v.) values of 0.17 micrograms/g and 0.83 micrograms/g, respectively. The major lethal toxin is the basic lethal phospholipase A, F6A, which accounts for approximately 13% of the venom protein and has a mol. wt of 21,000.
Some enzymatic activities and toxic properties of four samples of Ophiophagus hannah (king cobra) venom were investigated. There is little intraspecific variation in enzyme contents, protein composition and toxic properties of the venom. The venom does not exhibit hemolytic or edema-inducing activity but is characterized by an exceptionally high alkaline phosphomonoesterase activity. DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography of the venom indicate that the major lethal toxins are the low mol.wt, non-enzymatic basic proteins. Venom fractions exhibiting high enzymatic activities apparently do not play an important role in the lethality in mice of Ophiophagus hannah venom.
Snake venoms are complex mixtures of proteins and peptides that play vital roles in the survival of venomous snakes. As with their diverse pharmacological activities, snake venoms can be highly variable, hence the importance of understanding the compositional details of different snake venoms. However, profiling venom protein mixtures is challenging, in particular when dealing with the diversity of protein subtypes and their abundances. Here we described an optimized strategy combining a protein decomplexation method with in-solution trypsin digestion and mass spectrometry of snake venom proteins. The approach involves the integrated use of C18 reverse-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and nano-electrospray ionization tandem mass spectrometry (nano-ESI-LC-MS/MS).
The accumulation of paralytic shellfish toxins (PSTs) in contaminated shellfish is a serious health risk making early detection important to improve shellfish safety and biotoxin management. Capillary electrophoresis (CE) has been proven as a high resolution separation technique compatible with miniaturization, making it an attractive choice in the development of portable instrumentation for early, on-site detection of PSTs. In this work, capillary zone electrophoresis (CZE) with capacitively coupled contactless conductivity detector (C(4)D) and UV detection were examined with counter-flow transient isotachophoresis (tITP) to improve the sensitivity and deal with the high conductivity sample matrix. The high sodium concentration in the sample was used as the leading ion while l-alanine was used as the terminating electrolyte (TE) and background electrolyte (BGE) in which the toxins were separated. Careful optimization of the injected sample volume and duration of the counter-flow resulted in limit of detections (LODs) ranging from 74.2 to 1020 ng/mL for tITP-CZE-C(4)D and 141 to 461 ng/mL for tITP-CZE-UV, an 8-97 fold reduction compared to conventional CZE. The LODs were adequate for the analysis of PSTs in shellfish samples close to the regulatory limit. Intra-day and inter-day repeatability values (percentage relative standard deviation, n=3) of tITP-CZE-C(4)D and tITP-CZE-UV methods for both migration time and peak height were in the range of 0.82-11% and 0.76-10%, respectively. The developed method was applied to the analysis of a contaminated mussel sample and validated against an Association of Official Analytical Chemists (AOAC)-approved method for PSTs analysis by high performance liquid chromatography (HPLC) with fluorescence detection (FLD) after pre-column oxidation of the sample. The method presented has potential for incorporation in to field-deployable devices for the early detection of PSTs on-site.
An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.
1. Substrate specificity of purified king cobra (Ophiophagus hannah) venom L-amino acid oxidase was investigated. 2. The enzyme was highly specific for the L-enantiomer of amino acid. Effective oxidation of L-amino acid by the enzyme requires the presence of a free primary alpha-amino group but the alpha-carboxylate group is not as critical for the catalysis. 3. The enzyme was very active against L-Lys, L-Phe, L-Leu, L-Tyr, L-Tryp, L-Arg, L-Met, L-ornithine, L-norleucine and L-norvaline and moderately active against L-His, L-cystine and L-Ileu. Other L-amino acids were oxidized slowly or not oxidized. 4. The data suggest the presence of a side chain binding site in the enzyme, and that the binding site comprises at least five 'subsites': the hydrophobic subsites a, b and c; and the two 'amino' binding subsites d and e. Subsite b appears to be able to accommodate two methylene/methyl carbons.
A convenient acidimetric assay for phospholipase A using egg yolk suspension as substrate has been developed. The substrate mixture consists of 1 part egg yolk, 1 part 8.1 mM sodium deoxycholate, and 1 part 18 mM calcium chloride. Phospholipase A activity is measured by following the initial rate of pH change, which is linear between pH 8.0 and 7.75 and is proportional to enzyme concentration over a wide range. The assay is highly reproducible, with a coefficient of variation of 3%, and as sensitive as most established assays for phospholipase A. The assay uses inexpensive and easily available substrate and is simple to perform. It is particularly useful for monitoring phospholipase A activity in chromatography fractions.
The major hemorrhagin (termed hannahtoxin) of the venom of Ophiophagus hannah (king cobra) was purified to electrophoretic homogeneity by DEAE-Sephacel ion exchange chromatography, Sephadex G-200 gel filtration followed by a second DEAE-Sephacel chromatography. Proteolytic activity was associated with the hemorrhagic activity throughout the purification procedures. Hannahtoxin constituted approximately 2% of the crude venom. It had an isoelectric point of 5.3, a carbohydrate content of 12%, a mol. wt of 66,000 as determined by SDS-polyacrylamide gel electrophoresis and 63,000 as determined by gel filtration. It contains 1 mole of Zn per mole of protein. The minimum hemorrhage doses for hannahtoxin are 0.7 microgram and 75 micrograms, respectively, in rabbits and in mice. Hannahtoxin was not lethal to mice at a dose of 2 mg/kg (i.v.) but killed rabbits at doses above 0.18 mg/kg (i.v.). It liberated protein from rabbit glomerular basement membrane but not rat glomerular basement membrane. Treatment of the hemorrhagin with EDTA and 1,10-phenanthroline eliminated both the proteolytic and hemorrhagic activities completely.
The specificity and sensitivity of an indirect and two (an 'ordinary' and a 'rapid') double sandwich enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom were examined. The three assays were equally sensitive and the accuracy of the assays was not substantially affected by individual variation in the venom composition. The specificity of the assays was examined against 26 venoms from snakes of the families Viperidae and Elapidae. While the double sandwich ELISA procedures were sufficiently specific to be used in the clinical immunodiagnosis of C. rhodostoma bite in Malaysia, the indirect ELISA procedure exhibited extensive cross-reactivity with other Malaysian pit viper venoms. Attempts were made to improve the specificity of the indirect ELISA procedure for the quantitation of C. rhodostoma venom. A 'low ELISA cross-reactivity' venom fraction (termed VF52) was isolated from C. rhodostoma venom by repeated Sephadex G-100 gel filtration chromatography. The indirect ELISA procedure using antibodies to VF52 as immunoreagent showed an improvement in specificity. The use of the indirect ELISA procedure for the detection of C. rhodostoma antibodies was also examined and the results show that the assay was sufficiently specific to be used for retrospective diagnosis of C. rhodostoma bite in Malaysia, in particular when VF52 was used as the coating antigen.
The banded krait, Bungarus fasciatus is a medically important venomous snake in Asia. The wide distribution of this species in Southeast Asia and southern China indicates potential geographical variation of the venom which may impact the clinical management of snakebite envenomation. This study investigated the intraspecific venom variation of B. fasciatus from five geographical locales through a venom decomplexing proteomic approach, followed by toxinological and immunological studies. The venom proteomes composed of a total of 9 toxin families, comprising 22 to 31 proteoforms at varying abundances. The predominant proteins were phospholipase A2 (including beta-bungarotoxin), Kunitz-type serine protease inhibitor (KSPI) and three-finger toxins (3FTx), which are toxins that cause neurotoxicity and lethality. The venom lethality varied with geographical origins of the snake, with intravenous median lethal doses (LD50) ranging from 0.45-2.55 µg/g in mice. The Thai Bungarus fasciatus monovalent antivenom (BFMAV) demonstrated a dose-dependent increasing immunological binding activity toward all venoms; however, its in vivo neutralization efficacy varied vastly with normalized potency values ranging from 3 to 28 mg/g, presumably due to the compositional differences of dominant proteins in the different venoms. The findings support that antivenom use should be optimized in different geographical areas. The development of a pan-regional antivenom may be a more sustainable solution for the treatment of snakebite envenomation.
1. The two major phospholipase A2 enzymes (OHPLA-DE1 and OHPLA-DE2) of king cobra (Ophiophagus hannah) venom have been purified to electrophoretic homogeneity. 2. The isoelectric points of OHPLA-DE1 and OHPLA-DE2 were 3.81 and 3.89, respectively and the Mws were 14,000 and 15,000, respectively, as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-PAGE. 3. The enzymes were not lethal to mice at a dosage of 10 micrograms/g body wt by i.v. route. Both phospholipase A2 enzymes, however, exhibited moderate edema-inducing and anti-coagulant activities. 4. Bromophenacylation of the enzymes reduced the enzymatic activity drastically but did not affect the edema-inducing activity of the enzymes.
1. The L-amino acid oxidase, hyaluronidase, alkaline phosphomonoesterase, protease, phosphodiesterase, acetylcholinesterase, phospholipase A and 5'-nucleotidase activities of 47 samples of venoms from all the six species of cobra (Naja), including five subspecies of Naja naja, were examined. 2. The results demonstrated interspecific differences in the venom contents of phospholipase A, acetylcholinesterase, hyaluronidase and phosphodiesterase. These differences in venom enzyme contents can be used for the differentiation of species of the genus Naja. 3. Thus, our results revealed a correlation between the enzyme composition of venom and the taxonomic status of the snake at the species level for the genus Naja.
The Senegalese cobra, Naja senegalensis, is a non-spitting cobra species newly erected from the Naja haje complex. Naja senegalensis causes neurotoxic envenomation in Western Africa but its venom properties remain underexplored. Applying a protein decomplexation proteomic approach, this study unveiled the unique complexity of the venom composition. Three-finger toxins constituted the major component, accounting for 75.91% of total venom proteins. Of these, cardiotoxin/cytotoxin (~53%) and alpha-neurotoxins (~23%) predominated in the venom proteome. Phospholipase A2, however, was not present in the venom, suggesting a unique snake venom phenotype found in this species. The venom, despite the absence of PLA2, is highly lethal with an intravenous LD50 of 0.39 µg/g in mice, consistent with the high abundance of alpha-neurotoxins (predominating long neurotoxins) in the venom. The hetero-specific VINS African Polyvalent Antivenom (VAPAV) was immunoreactive to the venom, implying conserved protein antigenicity in the venoms of N. senegalensis and N. haje. Furthermore, VAPAV was able to cross-neutralize the lethal effect of N. senegalensis venom but the potency was limited (0.59 mg venom completely neutralized per mL antivenom, or ~82 LD50 per ml of antivenom). The efficacy of antivenom should be further improved to optimize the treatment of cobra bite envenomation in Africa.