Determination of molecular mass and partial peptide confirmation of short neurotoxins using chromatographic techniques and reverse phase HPLC.

The aim of present work was to investigate the purification of a novel protein (low molecular weight) from Indian cobra Naja naja by Cation exchange chromatography on CM-Sephadex C-25 and followed by Gelfiltration chromatography on Sephadex G-100. Fraction numbers 26, 27, and 28 were obtained from CM Sephadex C-25. From all the fractions, the protein concentration was calculated and it was applied onto the Sephadex G-100 Gelfiltration chromatography. Fraction No-11 obtained from Sephadex G-100 was used for the determination of molecular weight of the short neurotoxins by SDS-PAGE and Matrix- Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF). This SDS-PAGE is corresponding to the gel filtration chromatography which resolved a thick band of ~6-7 kDa proteins, and the MALDI-TOF resolved 6668.530, 7447.438 and 19928.929 Da. Further, this fraction was selected for preparative HPLC by using C18 column, in which two major peaks (retention time 30.793 and 32.846) were found. The peak with retention time of 30.793 was preferred for molecular mass determination by MALDI-TOF showed a single sharp peak of 6815.471 Da. It was digested with trypsin enzymatic cleavage, which explored approximately 26 peptides and their masses were renowned. The scores of all these 26 peptides were compared with online mascot analysis and BLAST sequence and it did not match with any other peptides and proteins. Among these 26 peptides, since only two peptides score as 886.648 and 943.690 Da were identical with short neurotoxin -1 from Naja oxiana, and short neurotoxin -3 from Naja mossambica. Moreover the 6815.471 Da protein was used for hemolytic activity and it did not induce RBC lysis. All this observations suggested that the newly purified protein is a short neurotoxin. This essential information will support us to find out the structural information of short neurotoxin for its application in anti-venom development, antitumor and also for analgesic effects.

Pattern of renal pathology in fatal envenomation by indian cobra (naja naja).

Deaths due to poisonous snakebite are a significant health related problem especially the rural heartland of in tropical countries. Renal involvement in snakebite is well documented especially so in bites by the Viperidae group. The Elapidae family consisting of cobra and kraits among other varieties are mainly considered neurotoxic. The venom of neurotoxic variety predominantly has direct depressing action on the respiratory center and neuromuscular junction. We investigated the renal changes at autopsy and histology of fatal cobra bites. This series included autopsy examination of 14 cases of fatal cobra bite in our hospital-based study. Dissected kidneys were sectioned, stained with hematoxylin & eosin stain and histological examination was done under light microscope. Five cases from head injury subject were used as control. The study reveals renal involvement in 64.28 %of fatal bites by Indian cobra (Naja naja) primarily considered neurotoxic. The major renal changes were tubular necrosis 1(7.14%), cortical necrosis 3 (21.42%) and interstitial nephritis 3(21.42%). This fact is worth giving due consideration during management and monitoring of cases of envenomation by cobra.

Characterization of an antibacterial peptide from indian cobra (Naja naja) venom

Due to the development of antibiotic resistance in microorganisms, antimicrobial peptides from natural sources have attracted attention in recent times. Several antimicrobial peptides have been isolated from a wide range of animal sources, particularly snake venoms. Naja naja venom showed antibacterial as well as direct and indirect hemolytic activities, and an antibacterial peptide was purified through gel permeation and ion exchange chromatography. Its molecular mass was 2491Da, which was determined using Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry and the amino acids sequence of the N-terminus was DEQSTHGAYVWKL. The purified peptide showed potent antibacterial activity against Gram-negative and Gram-positive bacterial strains like Escherichia coli, Pseudomonas aeruginosa and Vibrio cholerae, and Staphylococcus aureus, Enterococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Bacillus subtilis, respectively. The most potent activity was towards Gram-negative bacteria. Activity was retained at concentrations as low as 100µg/ml. Minimum inhibitory concentrations (MIC; in mg) of Naja Antibacterial Peptide (NAP) and known antibiotics against Gram-positive and Gram-negative bacteria were determined using microdilution susceptibility test in sterile 96-well microdilution plates. However, the peptide did not show direct or indirect hemolytic activity.(AU)