KINETIC AND THERMODYNAMIC STUDIES OF PURIFIED PROTEIN ISOLATED FROM EUPHORBIA TIRUCALLI LATEX

Authors

  • Tripti Sharma
  • Priyanka Dash
  • Debajyoti Das
  • Goutam Ghosh Department of pharmacognosy, School of Pharmaceutical Sciences ,Siksha 'o' Anusandhan University, Bhubaneswar

Abstract

 

 The kinetic and thermodynamic parameters of an isolated protein from the latex of Euphorbia tirucalli were studied in this work. The isolated protein was named as Euphorbian T and was found to possess proteolytic activity through milk clotting and caseinolytic digestion process. The electrophoresis analysis of purified Euphorbian T was carried out using sodium dodecyl sulfate-polyacrylamide gel. The aminoacids of Euphorbian T were appeared in different bands of 36.5, 39, 44 and 50 Kda. The effect of pH, temperature, and concentration of substrate on proteolytic activity of Euphorbian T was examined. The maximum proteolytic activity of this protein was obtained at pH 6. The temperature t(max) for maximum activity range of the enzyme was found to be 65°C. The Kinetics parameters i.e., Km and Vmax of the purified protein with milk and caesin as substrate, were estimated using Lineweaver–Burk plot and were found to be 4 mg/dl, 0.21 U/mg and 2 mg/dl, 1.03 U/mg, respectively. Thermodynamic constants i.e., activation energy (Ea), change in enthalpy (ΔH) and change in entropy (ΔS) and free energy changes (ΔG) of Euphorbian T for milk were calculated using Arrhenius plot and were found to be 1.58 kcal/mol, 1.54 kcal/mol, −10.46 cal mol/deg and 18.87 kcal/mol respectively, whereas with the substrate caesin, the activation energy (Ea), change in enthalpy (ΔH) and change in entropy (ΔS) and free energy changes (ΔG) were calculated as 1.12 kcal/mol, 1.08 kcal/mole, −16.24 cal mol/deg and 16.34 kcal/mol, respectively.

Keywords: Euphorbia tirucalli, Euphorbian T, Milk clotting, Caseinolytic.

Downloads

Download data is not yet available.

References

Sumantha A, Larroche C, Pandey A. Microbiology and industrial biotechnology of food-grade proteases: A perspective. Food Technol Biotechnol 2006;44(2):211-20.

Liggieri C, Obregon W, Trejo S, Priolo N. Biochemical analysis of a papain-like protease isolated from the latex of Asclepias curassavica L. Acta Biochim Biophys Sin (Shanghai)2009;41(2):154-62.

Vierstra RD. Proteolysis in plants: Mechanisms and functions. Plant Mol Biol 1996;32:275.

4. Prabha MN, Ramesh CK, Kuppast IJ, Mankani KI. Studies on anti-

inflammatory and analgesic activities of Euphorbia tirucalli latex. Int J Chem Sci 2008;6(4):1781-7.

Fridous AJ, Islam SN, Faruque AB. Antimicrobial activity of the leaves of Adhatoda vasica, Clatropis gigantean, Nerium odorum and Ocimum sanctum. Bangladesh J Bot 1990;227

Jyothi TM, Shankariah MM, Prabhu S, Lakshminarasu S, Srinivasa GM, Setty SR. Hepatoprotective and antioxidant activity of E. tirucalli. Iran J Pharmacol Ther 2008;7:25-30.

Valadares MC, Carrucha SG, Accorsi W, Queiroz ML. Euphorbia tirucalli L. modulates myelopoiesis and enhances the resistance of tumour-bearing mice. Int Immunopharmacol 2006;6:294-9.

Betancur-Galvis LA, Morales GE, Forero JE, Roldan J. Cytotoxic and antiviral activities of Colombian medicinal plant extracts of the Euphorbia genus. Mem Inst Oswaldo Cruz 2002;97(4):541-6.

Yadav R, Srivastava VK, Chandra R, Singh A. Larvicidal activity of latex and stem bark of Euphorbia tirucalli plant on the mosquito Culex quinquefasciatus. J Commun Dis 2002;34(4):264-9.

Wal A, Wal P, Gupta N, Vishnoi G, Srivastava RS. Medicinal value of Euphorbia tirucalli. Int J Pharm Biol Arch 2013;4(1):31-40.

Khan AQ, Malik A. A new macrocyclic diterpene ester from the latex of Euphorbia tirucalli. J Nat Prod 1990;53:728-31.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265-75.

Murata J, Satake M, Suzuki T. Studies on snake venom. XII. Distribution of proteinase activities among Japanese and Formosan snake venoms. J Biochem 1963;53:431-43.

Berridge NJ. An improved method of observing the clotting of milk containing rennin. J Dairy Res 1952;9:328-9.

Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987;166:368-79.

Lineweaver H, Burk D. The determination of enzyme dissociation constants. J Am Chem Soc 1934;56:658.

Rodriguez-Correa D, Dahlberg AE. Kinetic and thermodynamic studies of peptidyltransferase in ribosomes from the extreme thermophile Thermus thermophilus. RNA 2008;14(11):2314-8.

Khan M, Javed MM, Zahoor S, Ikram-ul-haq. Kinetics and thermodynamic study of urease extracted from soybeans. Biologia 2013;59(1):7-14.

Kumari M, Sharma A, Jagannadham MV. Religiosin B. A milk-clotting serine protease from Ficus religiosa. Food Chem 2012;131:1295-303.

Manohan D, Wai WC. Characterization of polyphenol oxidase in sweet potato (Ipomoea Batatas (L.). J Adv Sci Arts 2012;3(1):14-31.

Chanda I, Basu SK, Dutta SK, Das SR. Thermodynamic, kinetic and stability aspects of a purified protease from the latex of Plumeria rubra linn. Asian J Pharm Clin Res 2013;6(1):149-51.

Published

01-11-2014

How to Cite

Sharma, T., P. Dash, D. Das, and G. Ghosh. “KINETIC AND THERMODYNAMIC STUDIES OF PURIFIED PROTEIN ISOLATED FROM EUPHORBIA TIRUCALLI LATEX”. Asian Journal of Pharmaceutical and Clinical Research, vol. 7, no. 5, Nov. 2014, pp. 275-8, https://mail.innovareacademics.in/journals/index.php/ajpcr/article/view/2602.

Issue

Section

Original Article(s)

Most read articles by the same author(s)