Isolation and characterization of snske venom proteins and peptides from viperideride (bitis arietans) and elapidae (NAJA) melanoleuca naja ashei dendroaspis angusticeps) families in Kilifi County.

Abstract

Snake venom comprises highly complex mixture of protein and peptides. Kenya is inhabitated by many species of venomous snakes from both Viperidae and Elapidae families. This study aimed to isolate and biochemically characterize snake toxins from members of Viperidae (Bitis arietans) as well as Elapidae (Naja melanoleuca, Naja ashei and Dendroaspis angusticeps) from Kilifi County. This cross sectional study was done on venom samples extracted from eight adult snake specimens, both male and female representing each species. Two-dimensional gel Electrophoresis method was used to analyze the samples and later stained by silver staining technique. Two-dimensional gels protein spots were analyzed using verified empirical isoelectric points and molecular weights generated in comparison with the incorporated protein standards and these data used as parameters in the protein identification program, TagIdent. This program allowed the identification of whole protein present in the 2D gel spot using its estimated pI, molecular masses, keywords and species (or group of species) of interest. The estimated isoelectric points and molecular weights data generated enabled further determination of the physical and biochemical characteristics of the venom protein toxins identified earlier. This study findings suggest that the venom protein toxins from Elapidae families contained very low molecular weights of approximately 6000 daltons to 14,400 daltons, while on the other hand, the Viperidae family had spotting at the molecular mass ranges of 14,400 daltons to 36,500 daltons. Overall, the venom toxin‘s isoelectric point ranged from pI 4 to pI 8 and molecular mass of 7515 daltons to 13473 daltons. This study identified the five snake venom proteins, four from Naja (Boulengrina) subfulva formerly melanoleuca and one from Dendroaspis angusticeps in a two-dimensional gel electrophoresis (2D) after an in-depth computer search from Swiss-Prot protein database. They were {P00599}- Basic phospholipases A2 I, with amino acid chain length of 1-118, pI=7.55, Mw:13473 daltons; {P00600}-Acidic phospholipases A2 DE-II, chain:1-119, pI=6.82, Mw: 13427 daltons; {P00601}–Acidic phospholipases A2 DE-III, chain:1-119, pI=6.27, Mw: 13360 daltons); {P01383}- Long c h a i n neurotoxin 1 with 1-71 amino acid residues, 8056 daltons, pI=8.73 and {P81030}- Three-finger muscarinic toxin1 with 1-66 amino acids, 7518 daltons, pI=8.48. This study finding suggests that the five identified venom protein toxins are thermodynamically stable and highly hydrophilic molecules. However, five 2D-gel proteins spots turned out to be negative and/or undetermined which may represent some of the previously undescribed toxins that are yet to be deposited in protein databases and requires further in-gel enzymatic digestion, liquid chromatography mass spectrometry and western blot analysis to confirm their presence in the venom sample. Therefore, further studies are needed to confirm the presence and identities of these unknown proteins to enable better understanding of their structure―function relationships and application as vaccine immunotherapeutic agents aiming to address the public health problem of snakebite disabilities and improve human health in the affected populations.