The snake venomic of Crotalus durissus terrificus was analyzed by 2-D and 1-D electrophoresis and subsequent MS/MS and enzymatic assays. The venomic of the South American rattlesnake comprises toxins from seven protein families: phospholipases A2, serine proteinases, ecto-5′-nucleotidases, metalloproteinases, nerve growth factors, phosphodiesterases, and glutaminyl cyclase. The venom toxin composition correlates with the clinical manifestation of the crotalinae snake bites and explains pathological effects of the venom such as neurotoxicity, systemic myonecrosis, hemostatic disorders, myoglobinuria, and acute renal failure. The vast majority of toxins are potentially involved in neurotoxicity, myotoxicity, and coagulopathy. The predominant venom components are neurotoxic phospholipases A2 and serine proteinases. The venom is a rich source of 5′-nucleotidases (7.8% of the identified toxins) inducing hemostatic disorders. Analysis of the venom protein composition provided a catalogue for secreted toxins. The venomic composition of Crotalus d. terrificus and venom gland transcriptome of the synonymous subspecies Crotalus d. collilineatus show differences in the occurrence of protein families and in the abundance of toxins. Some of the venom components identified by the proteomic analysis were not reported in the transcriptome of the Crotalus d. collilineatus venom gland. Enzymatic activities of the Crotalus d. terrificus venom were determined and correlated with the proteomic composition.Keywords: 2-D electrophoresis; Crotalus durissus terrificus; electrospray mass spectrometry; snake venomic;

Controlled navigation in the phase diagram of protein crystallization and probing by advanced Dynamic Light Scattering (DLS) technology provided new information and more insight on the early processes during the nucleation process. The observed hydrodynamic radius distribution pattern clearly reveals a two-step mechanism of nucleation and the occurrence of liquid dense protein clusters, which were verified by transmission electron microscopy. The growth kinetics of these protein clusters, forming distinct radii fractions, is analyzed in real time. Further, the data confirmed that critical nuclei show a distinctly different radius distribution than the liquid dense clusters. The data and results provide experimental evidence that during nucleation, a formation of distinct liquid clusters with high protein concentration occur prior to a transition to crystal nuclei by increasing the internal structural order of these clusters, subsequently.

The successful crystallization of proteins is important because their molecular three-dimensional structures can be obtained through X-ray diffraction when in their crystalline form. Investigation of the crystallization process is beneficial for this purpose. We have reported that protein crystallization is sensitive to audible sound, which is commonly present but is often ignored. Here we investigate the effect of audible sound parameters, especially frequency, on a protein crystallization. We show a significant facilitation of protein crystallization using 5000 Hz audible sound, possible mechanism was also tried to be clarified. Suitably controlled audible sound can be beneficial for promoting protein crystallization. Therefore, audible sound can be used as a simple tool to promote protein crystallization. In addition, the processing of other types of materials may also be affected by audible sound.

Myotoxicity and membrane damage play a central role in the life-threatening effects of the viper envenomation. Myotoxins are an important part of the viper venomics. A Ser49 PLA2-like myotoxin from the venom of Vipera ammodytes meridionalis, the most venomous snake in Europe, was crystallized and its three-dimensional structure determined. The toxin is devoid of phospholipolytic activity. The structure demonstrates a formation of dimers. In the dimers functionally important peptide segments, located on the protein surface, point in the same direction which can strengthen the pharmacological effect. This supports the hypothesis about the physiological importance of the toxin oligomerization for the myotoxicity and membrane damage. The crystallographic model revealed that the structural determinants of myotoxicity (a positively charged C-terminal region and a hydrophobic knuckle) are fully exposed on the protein surface and accessible for interactions with target membranes. Distortion of the catalytic site region explains the absence of enzymatic activity. The structure reveals anion-binding sites which can be considered as possible sites of interactions of the toxin with a negatively charged membrane surface. The high structural similarity of the Ser49 myotoxin and Asp49 PLA2 from the same venom suggests an evolutionary relationship: probably, the Ser49 myotoxin is a product of evolution of the catalytically active phospholipase A2. The first toxin lost the enzymatic activity which is not necessary for the myotoxicity but preserved the cytotoxicity and membrane damaging activity as important components of the venom toxicity.

The venom composition of Pseudechis australis, a widely distributed in Australia reptile, was analyzed by 2-DE and mass spectrometric analysis. In total, 102 protein spots were identified as venom toxins. The gel is dominated by horizontal trains of spots with identical or very similar molecular masses but differing in the pI values. This suggests possible post-translational modifications of toxins, changing their electrostatic charge. The results demonstrate a highly specialized biosynthesis of toxins destroying the hemostasis (P−III metalloproteases, SVMPs), antimicrobial proteins (l-amino acid oxidases, LAAOs, and transferrin-like proteins, TFLPs), and myotoxins (phospholipase A2s, PLA2s). The three transferrin isoforms of the Australian P. australis (Elapidae snake) venom are highly homologous to the body transferrin of the African Lamprophis fuliginosus (Colubridae), an indication for the recruitment of body transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey. Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-5′-nucleotidases (5′-NTDs), nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymatic activities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, and biotechnological purposes.

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.

The venom proteome of Bothrops alternatus, a venomous snake widespread in South America, was analyzed by 2-D electrophoresis followed by mass spectrometric analysis and determination of enzymatic activities. The venomic composition revealed that metallo- and serine proteinases play primary roles in the pathogenesis of the envenomation by this pitviper. The identified 100 venom components with molecular masses from 10 to 100 kDa belong to six protein families: metalloproteinases, serine/thrombin-like proteinases, phospholipases A2, L-amino acid oxidases, disintegrins and thrombin inhibitors. Metalloproteinases predominate and belong exclusively to the P-III class including the most potent hemorrhagic toxins. They represent 50% of all identified proteins. Two isoforms were identified: homologous to jararhagin, a hemorrhagic toxin, and to beritractivase, a nonhemorrhagic and pro-coagulant metalloproteinase. The B. alternatus venom is a rich source of proteins influencing the blood coagulation system with a potential for medical application. The isoelectric points of the components are distributed in the acidic pH range (the pI values are between 4 and 7) and no basic proteins were detected.

Api m 7 is one of the major protease allergens of the honeybee venom. It consists of a serine protease-like (SPL) and a CUB domain. The knowledge about the structure and function of Api m 7 is limited mainly to its amino acid sequence. Three-dimensional models of the two structural domains were constructed using their amino acid sequences and the crystallographic coordinates of prophenoloxidase-activating factor (PPAF-II) as a template for the SPL domain and the coordinates of porcine spermadhesin PSP-II for the CUB domain. The structural organization of Api m 7 suggests that the CUB domain is involved in interactions with natural substrates while the SPL domain probably activates zymogens. IgE epitopes and antigenic sites were predicted. Api m 7 shows structural and functional similarity to the members of the PPAF-II family. Possible substrates, function and evolution of the enzyme are discussed in the paper.