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Cobra venom (Naja kaouthia) contains a toxin called α-cobratoxin (α-Cbtx). This toxin is a natural protein containing 71 amino acids (MW 7821 Da) with a reported analgesic potency greater than morphine. In 2007, in USA, this substance was found in the barns of a thoroughbred trainer and since then till date, the lack of a detection of this molecule has remained a recurring problem for the horseracing industry worldwide. To solve this problem, the first method for the detection of α-cobratoxin in equine plasma has now been developed. Plasma sample (3 mL) was treated with ammonium sulfate at the isoelectric point of α-Cbtx, and the pellet was dissolved in a phosphate buffer and mixed with methanol for precipitation. The supernatant was then concentrated prior to its extraction on WCX SPE cartridges. The eluate was concentrated with two consecutive filtration steps before the trypsin digestion. The samples were analyzed using a LC-MS/MS Q Exactive instrument at 70,000 resolution on the product ions of the doubly charged precursor of the target peptide (24TWCDAFCSIR33). The method was validated (n = 18) at 5 μg/L (640 pmol/L) according to the Association of Official Racing Chemists (AORC) requirements. The lower limit of detection was 1 μg/L (130 pmol/L). The present method has made it possible for us to confirm the presence of α-Cbtx in a horse plasma sample 24 h post the administration of α-Cbtx. Thus, the present method provides the first sensitive, specific, and reliable analytical method to confirm the presence of α-Cbtx in equine plasma. [ABSTRACT FROM AUTHOR]

The major cardiotoxin from Taiwan cobra (CTX A3) is a pore forming β-sheet polypeptide that requires sulfatide (sulfogalactosylceramide, SGC) on the plasma membrane of cardiomyocytes for CTX-induced membrane leakage and cell internalization. Herein, we demonstrate by fluorescence spectroscopic studies that sulfatides induce CTX A3 oligomerization in sulfatide containing phosphatidylcholine (PC) vesicles to form transient pores with pore size and lifetime in the range of about 30 Å and 10-2 s, respectively. These values are consistent with the CTX A3-induced conductance and mean lifetime determined previously by using patch —clamp electrophysiological experiments on the plasma membrane of H9C2 cells. We also derived the peripheral binding structural model of CTX A3—sulfatide complex in sulfatide containing PC micelles by NMR and molecular docking method and compared with other CTX A3—sulfatide complex structure determined previously by X-ray in membrane-like environment. The NMR results indicate that sulfatide head group conformation changes from a bent shovel (–sc/ap) to an extended (sc/ap) conformation upon initial binding of CTX A3. An additional global reorientation of sulfatide molecule is also needed for CTX A3 dimer formation as inferred by the difference between the X-ray and NMR complex structure. Since the overall folding of CTX A3 molecules remained the same, sulfatide in phospholipid bilayer is proposed to play an active role by involving its local and global conformational changes to promote both the oligomerization and reorientation of CTX A3 molecule for its transient pore formation and cell internalization. [ABSTRACT FROM AUTHOR]

Phospholipase A2 (PLA2)-catalyzed hydrolysis at the sn-2 position of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in optically trapped liposomes is monitored in situ using confocal Raman microscopy. Individual optically trapped liposomes (0.6 μm in diameter) are exposed to PLA2 isolated from cobra (Naja naja naja) venom at varying enzyme concentrations. The relative Raman scattering intensities of C—C stretching vibrations from the trans and gauche conformers of the acyl chains are correlated directly with the extent of hydrolysis, allowing the progress of the reaction to be monitored in situ on a single vesicle. In dilute vesicle dispersions, the technique allows the much higher local concentration of lipid molecules in a single vesicle to be detected free of interferences from the surrounding solution. Observing the local composition of an optically trapped vesicle also allows one to determine whether the products of enzyme-catalyzed hydrolysis remain associated with the vesicle or dissolve into solution. The observed reaction kinetics exhibited a time lag prior to the rapid hydrolysis. The lag time varied inversely with the enzyme concentration, which is consistent with the products of enzyme-catalyzed lipid hydrolysis reaching a critical concentration that allows the enzyme to react at a much faster rate. The turnover rate of membrane-bound enzyme determined by Raman microscopy during the rapid, burst-phase kinetics was 1200 s-1. Based on previous measurements of the equilibrium for PLA2 binding to lipid membranes, the average number of enzyme molecules responsible for catalyzing the hydrolysis of lipid on a single optically trapped vesicle is quite small, only two PLA2 molecules at the lowest enzyme concentration studied. [ABSTRACT FROM AUTHOR]

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 fl-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. [ABSTRACT FROM AUTHOR]

Studies the structure and dynamics of the major P-type cardiotoxins (CTX) from Taiwan cobra, CTX A3 by a comprehensive nuclear magnetic resonance (NMR) analysis involving [sup 1]H NOESY/ROESY, [sup 13C{[sup 1]H} NOE/T[sub 1] relaxation and [sup 17]O triple-quantum filtered NMR. Role of the water-binding loop in toxin binding to biological membranes and in their cytotoxicity; Exchange rate of the bound water in the loop.

Describes a complete amino acid sequence of kaouthiagin, a cobra venom metalloproteinase with disintegrin-like sequences. Primary structure of kaouthiagin; Effects of inactivated kaouthiagin and synthetic peptides of disintegrin-like domains on agonist-induced platelet aggregation.

Reports that cardiotoxin analogue III and cobrotoxin isolated from the Taiwan cobra (Naja naja atra) venom are structurally homologous, small molecular weight, all-beta-sheet proteins, cross-linked by disulfide bonds at identical positions. Comparison of the conformational stabilities of these toxins; Chemical shifts and amide proton exchange kinetics.

Examines the role of aromatic residues in the interfacial catalysis of Naja naja atra acidic phospholipase A2 (PLA2). Expression of recombinant PLA2 and mutants; Properties of PLA2 mutants towards polymerized mixed liposomes; Membrane binding affinity of wild type and PLA2 mutants; Penetration of wild type and mutants in the monolayer.

Examines the structural stability of cardiotoxin (CTX) analogue III from the Taiwan cobra by hydrogen-deuterium (H/D) exchange kinetics. Relationship between H/D exchange and structure; Protection factors and time constants of refolding residues in CTX III.

Studies the crystal structure of cardiotoxin V from Taiwan cobra venom (CTX A5). Three-dimensional structures of snake cardiotoxins; Conformational changes in loop structure; Novel membrane-binding motif in the three-finger loops of P-type cardiotoxin; Disparity in the structure of CTX A5; Isolation and crystallization of CTX A5.

Studies the role of acidic amino acid residues in the structural stability of snake cardiotoxins (CTX). Nuclear magnetic resonance (NMR) and circular dichroism (CD) studies of effect of acidic pH on CTX A5; Comparative studies of CTX homologues; Structural similarities of CTX homologues near C-terminal.

Studies conformational change and inactivation of membrane phospholipid-related activity of cardiotoxin (CTX A5) V from Taiwan cobra, Naja anaj atra, venom at acidic pH. Binding of CTX to phosphatidylcholine monolayers; pH-dependence of local conformational change of CTX.