Nature endowed snakes with a lethal secretion known as venom, which has been fine-tuned over millions of years of evolution. Snakes utilize venom to subdue their prey and to survive in their natural habitat. Venom is known to be a very poisonous mixture, consisting of a variety of molecules, such as carbohydrates, nucleosides, amino acids, lipids, proteins and peptides. Proteins and peptides are the major constituents of the dry weight of snake venoms and are of main interest for scientific investigations as well as for various pharmacological applications. Snake venoms contain enzymatic and non-enzymatic proteins and peptides, which are grouped into different families based on their structure and function. Members of a single family display significant similarities in their primary, secondary and tertiary structures, but in many cases have distinct pharmacological functions and different bioactivities. The functional specificity of peptides belonging to the same family can be attributed to subtle variations in their amino acid sequences. Currently, complementary tools and techniques are utilized to isolate and characterize the peptides, and study their potential applications as molecular probes, and possible templates for drug discovery and design investigations.

Snake venom is a source of many pharmacologically important molecules. Agkistrodon bilineatus commonly known as Cantil, is spread over Central America particularly in Mexico and Costa Rica. From the venom of Agkistrodon bilineatus we have isolated and characterised six hypotensive peptides, and two bradykinin inhibitor peptides. The IC-50 value of four synthesized peptides was studied, towards angiotensin converting enzyme, in order to study the structure-function relationship of these peptides. The purification of the peptides was carried out by size exclusion chromatography, followed by reverse phase chromatography. Sequences of all peptides were determined applying MALDI-TOF/TOF mass spectrometry. These hypotensive peptides bear homology to bradykinin potentiating peptides and venom vasodilator peptide. The peptide with m/z 1355.53 (M + H)+1, and the corresponding sequence ZQWAQGRAPHPP, we identified for the first time. A precursor protein containing a fragment of this peptide was reported at genome level, (Uniprot ID P68515), in Bothrops insularis venom gland. These proline rich hypotensive peptides or bradykinin potentiating peptides are usually present in the venom of Crotalinae, and exhibit specificity in binding to the C domain of somatic angiotensin converting enzyme. Four of these hypotensive peptides, were selected and synthesized to obtain the required quantity to study their IC50 values in complex with the angiotensin converting enzyme. The peptide with the sequence ZLWPRPQIPP displayed the lowest IC50 value of 0.64 μM. The IC50 value of the peptide ZQWAQGRAPHPP was 3.63 μM. The canonical snake venom BPPs classically display the IPP motif at the C-terminus. Our data suggest that the replacement of the highly conserved hydrophobic isoleucine by histidine does not affect the inhibitory activity, indicating that isoleucine is not mandatory to inhibit the angiotensin converting enzyme. The evaluation of IC 50 values show that the peptide with basic pI value exhibits a lower IC 50 value.

Latest advancement of omics technologies allows in-depth characterization of venom compositions. In the present work we present a proteomic study of two snake venoms of the genus Naja i.e., Naja naja (black cobra) and Naja oxiana (brown cobra) of Pakistani origin. The present study has shown that these snake venoms consist of a highly diversified proteome. Furthermore, the data also revealed variation among closely related species. High throughput mass spectrometric analysis of the venom proteome allowed to identify for the N. naja venom 34 protein families and for the N. oxiana 24 protein families. The comparative evaluation of the two venoms showed that N. naja consists of a more complex venom proteome than N. oxiana venom. Analysis also showed N-terminal acetylation (N-ace) of a few proteins in both venoms. To the best of our knowledge, this is the first study revealing this posttranslational modification in snake venom. N-ace can shed light on the mechanism of regulation of venom proteins inside the venom gland. Furthermore, our data showed the presence of other body proteins, e.g., ankyrin repeats, leucine repeats, zinc finger, cobra serum albumin, transferrin, insulin, deoxyribonuclease-2-alpha, and other regulatory proteins in these venoms. Interestingly, our data identified Ras-GTpase type of proteins, which indicate the presence of extracellular vesicles in the venom. The data can support the production of distinct and specific anti-venoms and also allow a better understanding of the envenomation and mechanism of distribution of toxins. Data are available via ProteomeXchange with identifier PXD018726.