PURIFICATION AND BIOCHEMICAL CHARACTERIZATION OF L-AMINO ACID OXIDASE FROM WESTERN REGION INDIAN COBRA (NAJA NAJA) VENOM
Keywords:
Indian Cobra (Naja naja), Venom, L amino acid oxidase-LAAOAbstract
Objective: Purification and biochemical characterization of LAAO from western region Indian Cobra (Naja naja) venom.
Methods: LAAO was purified from Indian cobra (Naja naja) venom using sequential chromatography on Sephadex G-75 gel filtration followed by Ion exchange on CM-Sephadex C-25 column. Biochemical characterization viz., pH, Temperature, Km and Vmax were determined. Molecular weight of LAAO was determined by electrophoresis. Inhibition of LAAO from cold water extracts of Curcuma zedoria, Curcuma ceasia, Curcuma aromatic, Curcuma longa, Curcuma amada, Cucumis sativus and Benincasa hispida was done.
Results: Purified LAAO showed the single band on non reducing SDS-PAGE with approximate molecular weight of 65 kDa. Further biochemical characterization revealed that, LAAO from Naja naja (western region) has an optimum pH of 7.0 and is stable at room Temperature upto 37 °C and showed an optimum enzyme concentration of 4µg/ml and Km 134.1µM and Vmax is 21.87 U/min. Cold water extract of Curcuma ceasia, Curcuma aromatic and Benincasa hispida showed comparatively significant inhibition of LAAO.
Conclusion: LAAO has promising therapeutic prospects because of its effects on various biological functions. Variation in snake species and their geographical distribution also contributes to the venom properties such as composition, toxicity level, pharmacological and biological activities. The significant difference in protein profiling leads to substantial lethality among different geographical regions. Thus in this study Indian Cobra (Naja naja) venom from the western region of India was subjected to purification of LAAO and biochemical characterization.
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Mohapatra B, Warrell, David A, Suraweera, Wilson, Bhatia, et al. Snakebite mortality in india: a nationally representative mortality survey. PLoS Neglected Trop Dis 2011;5:e10-8.
Narendra Sharath Chandra JN, Ponnappa KC, Sadashiva CT, Priya BS, Nanda BL, Veerabasappa T Gowda, et al. Chemistry and structural evaluation of different phospholipase A2 inhibitors in arachidonic acid pathway mediated inflammation and snake venom toxicity. Curr Top Med Chem 2007;7:787-800.
Kumar JR, Basavarajappa BS, Arancio O, Aranha I, Gangadhara NS, Yajurvedi HN, et al. Isolation and characterization of Reprotoxinâ€, a novel protein complex from daboia russelii snake venom. Biochimie 2008;90:1545-59.
Kasturi S, T Veerabasappa Gowda. Purification and characterization of a major phospholipase A2 from Russell's viper (Vipera russelli) venom. Toxicon 1989;27:229-37.
Du, Xiao-Yan, Clemetson, Kenneth J. Snake venom L-amino acid oxidases. Toxicon 2002;40:659-65.
Huang CL. Venoms: chemistry and molecular biology. J Pharm Sci 1978;67:893-3.
Coudert, Michele, Vandecasteele, Jean-Paul. Characterization and physiological function of a soluble L-amino acid oxidase in Corynebacterium. Arch Microbiol 1975;102:151-3.
Yang H, Johnson PM, Ko KC, Kamio M, Germann MW, Derby CD, et al. Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid oxidase from ink of the sea hare Aplysia californica. J Exp Biol 2005;208:3609-22.
Kusakabe H, Kodama K, Kuninaka AA. New antitumor enzyme, L-lysine alpha-oxidase from Trichoderma viridea. Purification and enzymological properties. J Biol Chem 1998;255:976-81.
Kitani Y, Tsukamoto C, Zhang G. Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli. FEBS J 2007;274:125-36.
Massey, Vincent, Curti B. On the reaction mechanism of crotalus adamanteus L-Amino acid oxidase. J Biol Chem 1967;242:1259-64.
Raghu Ram Achar, Venkatesh BK, Sharanappa P, Priya BS, S Nanjunda Swamy. Evidence for peroxidase activity in Caralluma umbellata. Appl Biochem Biotechnol 2014;173:1955-62.
Wiese AG, Pacifici RE, Davies KJA. Transient adaptation to oxidative stress in mammalian cells. Arch Biochem Biophys 1995;318:231-40.
Naumann GB, Liliana F Silva, Luciana Silva. Cytotoxicity and inhibition of platelet aggregation caused by an LAAO from Bothrops leucurus venom. Biochim Biophys Acta 2011;1810:683-94.
Stábeli, Rodrigo G. Platelet aggregation and antibacterial effects of an L-amino acid oxidase purified from Bothrops alternatus snake venom. Bioorg Med Chem 2004;12:2881-6.
Sarkar, Priyabrata, Tothill, Ibtisam E, Setford, Steven J, et al. Screen-printed amperometric biosensors for the rapid measurement of L-and D-amino acids. Analyst 1999;124:865-70.
Ande, Sudharsana Rao K, Phaneeswara Rao D, Sigrid M, Michael M, Peter G, et al. Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom. Apoptosis 2006;11:1439-51.
Shashidharamurthy R, Jagadeesha DK, Girish KS, Kemparaju K. Variation in biochemical and pharmacological properties of Indian cobra (Naja naja naja) venom due to geographical distribution. Mol Cell Biochem 2002;229:93-101.
Mukherjee AK, Maity CR. The composition of Naja naja venom samples from three districts of west bengal, India. Comp Biochem Physiol Part A: Mol Integr Physiol 1998;119:621-7.
Shashidharamurthy R, Kemparaju K. Region-specific neutralization of Indian cobra (Naja naja) venom by polyclonal antibody raised against the eastern regional venom: a comparative study of the venoms from three different geographical distributions. Int Immunopharmacol 2007;7:61-9.
Bradford, Marion M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.
Berger R. Methods of enzymatic analysis (3rd Edition). Volume II Samples, Reagents, Assessment of Resultsâ€. Weinheim/Deerfield Beach, Florida/Basel: Verlag Chemie 1983. 539 S., 37 Abb, 21 Tab., 215 DM (wenn alle Bände), 245 DM (Einzelband). Acta Biotechnol 1984;4:332-2.
Davis, Baruch J. Disc electrophoresis-II method and application to human serum proteins. Ann N Y Acad Sci 1964;121:404-27.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-5.
Shivalingu BR, Vivek HK, Zohara N, Priya BS, S Nanjunda Swamy. Comparative analysis of procoagulant and ï¬brinogenolytic activity of crude protease fractions of turmeric species. J Ethnopharmacol 2015;172:261–4.
Zohara Nafeesa, Shivalingu BR, Vivek HK, Priya BS, S Nanjunda Swamy. Exploring a new serine protease from Cucumis sativus L. Appl Biochem Biotechnol Part A 2015;175:2787-94.
Keerthy HK, Garg M, Mohan Chakrabhavi D, Madan, Vikas, Kanojia D, et al. Synthesis and characterization of novel 2-amino-chromene-nitriles that target Bcl-2 in acute myeloid leukemia cell lines. PLoS One 2014;9:e107-18.
Dineshkumar P, Muthuvelan B. Isolation and purification of l-amino acid oxidase from indian cobra Naja naja. Curr Res J Biol Sci 2011;3:6-11.