Determination of molecular mass and partial peptide confirmation of short neurotoxins using chromatographic techniques and reverse phase HPLC.
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Date
2011-10
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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.
Description
Keywords
Snake venom, Indian cobra Naja naja, Peptide mapping, Short neurotoxin
Citation
Mitra Bhaskar, Katiyar Monica, Kumar Amod, Palanichamy V. Determination of molecular mass and partial peptide confirmation of short neurotoxins using chromatographic techniques and reverse phase HPLC. International Journal of Applied Biology and Pharmaceutical Technology. 2011 Oct-Dec; 2(4): 150-157.