ABSTRACT
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.