Partial Purification and Characterization of Lipoxygenase Enzyme From Bovine Liver, Effects of Salicylic Acid and Some Flavons on the Enzyme

Abstract

Lipoxygenase (EC 1.13.11.34; LOX) is a non-hem iron-bearing dioxygenase which converts oxygen into fatty acids containing two or more unsaturated bonds and converts them into fatty acid hydroperoxides. Lipoxygenase activity is described in developed plants, in mammals, in echolalia, in fungi, in bacteria. LOX 5, LOX 8, LOX 12 and LOX 15, the four-headed isoenzymes of LOX enzymes are commonly found in animal tissues. In this study, LOX enzyme from bovine liver was partially purified by precipitation of ammonium sulphate at 30-50% saturation. Characterization studies were carried out and the optimum ionic strength value was determined as 0.5 mM, optimum pH value was pH 4.0 and optimum temperature value was 30 °C. The inhibitory effect of salicylic acid, eupatorin, eupatilin and gardenin A on LOX enzyme was investigated. Inhibitory effect of salicylic acid, eupatorin, eupatilin and gardenin A on LOX enzyme was investigated. IC50 values were calculated as 1.43 μM for salicylic acid, 0.46 μM for eupatorin, 0.15 μM for eupatilin and 5.31 μM for gardenin A.

Keywords

• Lipoxygenase
• partial purification
• characterization
• inhibition

Lipoksigenaz Enziminin Sığır Karaciğerinden Kısmi Saflaştırılması, Karakterizasyonu, Salisilik Asitin ve Bazı Flavonların Enzim Üzerine Etkileri

Abstract

Lipoksigenaz (EC 1.13.11.34.; LOX ) enzimleri; iki ya da daha fazla doymamış bağ bulunduran yağ asitlerine oksijen katarak oksitleyen, yapısında hem grubu olmayan demir taşıyıcı dioksigenazlardır. Gelişmiş bitkilerde, memelilerde, ökoryatik alglerde, fungilerde, bazı bakterilerde de tanımlanmıştır. Hayvan dokularında, LOX 5, LOX 8, LOX 12 ve LOX 15 izoenzimlerinin bulunduğu doğrulanmıştır. Bu çalışmada, sığır karaciğerinden LOX enzimi % 30-50 doygunluk aralığında amonyum sülfat çöktürmesiyle kısmi saflaştırıldı. Karakterizasyon çalışmaları yapılarak, optimum iyonik şiddet değeri 0.5 mM, optimum pH değeri 4.0 ve optimum sıcaklık değeri 30 °C olarak belirlendi. LOX enzimi aktivitesi üzerinde salisilik asit, eupatorin, eupatilin ve gardenin A maddelerinin inhibisyon etkisini araştırmak üzere 5 farklı inhibitör konsantrasyonunda aktivite ölçümü yapılarak % Aktivite –[I] grafikleri çizildi, IC50 değerleri bulundu. Salisilik asit için 1.43 μM, eupatorin için 0.46 μM, eupatilin için 0.15 μM ve gardenin A için 5.31 μM olarak belirlendi.

Keywords

• Lipoksigenaz
• kısmi saflaştırma
• karakterizasyon
• inhibisyon

References

1. Aparoy P, Reddy RNL, Gurupsarad MR, Reddy P, Reddanna, 2008. Homology modeling of 5-lipoxygenase and hints for better inhibitor design. Journal of Computer-Aided Molecular Design, 22: 611–619.
2. Brash AR,2015. Catalysis, and acquisition of substrate lipoxygenases: occurrence, functions. Journal of Biological Chemistry, 274: 23679-23682.
3. Charlier C, Michaux C, 2003. Dual inhibition of cyclooxygenase-2 (COX- 2) and 5-lipoxygenase (5-LOX) as a new strategy to provide safer non-steroidal anti-inflammatory drugs. European Journal of Medicinal Chemistry, 38: 645-659.
4. Dobrian AD, Lieb DC, Cole BK, Taylor-Fishwick DA, 2011. Functional and pathological roles of the 12- and 15-lipoxygenases. Progress in Lipid Research., 50: 115-131.
5. Funk CD, 1996. The molecular biology of mammalian lipoxygenases and the quest for eicosanoid functions using lipoxygenase-deficient mice. Biochimica et Biophysica Acta.,1304: 65-84.
6. Iversen L, Kragbella K, 2000. Arachidonic acid metabolism in skin health and disease. Prostaglandins Other Lipid Mediators, 63: 25-42.
7. Jaenicke, L, 1984 Einführung in die Praxis des Biochemikers, Institut für Biochemie, Universität zu Köln, Druck: Photostelle der Universitäts- und Stadtbibliothek Köln, 268-272
8. Juntachote T, Berghofer E, 2005. Antioxidant properties and stability of ethanolic extracts of Holy basil and Galangal. Food Chemistry, 92: 193-202.

9. Kelavkar UP, Cohen C, Kamitani H, Eling TE, Badr KF, 2000. Concordant induction of 15-lipoxygenase-1 and mutant p53 expression in human prostate adenocarcinoma: correlation with Gleason stating. Carcinogenesis, 21:1777-1787.
10. Khan H, Saeed M, Muhammad N, Gaffar R, Gul F, Raziq N, 2015. Lipoxygenase and urease inhibition of the aerial parts of the Polygonatum verticillatum. Toxicol Ind Health, ( 8):758-63.
11. Khanna S, Roy S, Ryu H, Bahadduri P, Swaan PW, Ratan RR, Chandan K, Sen CK, 2003. Molecular basis of vitamin E action. Journal of Biological Chemistry, 278: 43508-43515.
12. Kuo JM, Hwang A, Yeh DB, Pan MH, Tsai ML, Pan BS, 2006. Lipoxygenase from banana leaf: purification and characterization of an enzyme that catalyzes linoleic acid oxygenation at the 9-position. J Agric Food Chem., 54(8):3151-6.
13. Lu X, Zhang J, Liu S, Zhang D, Xu Z, Wu J, Li J, Du G, Chen J, 2013. Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli. Appl Microbiol Biotechnol.;97(13):5793-800.
14. Min, S, Min SK, Zhang QH, 2003. Inactivation Kinetics of Tomato Juice Lipoxygenase by Pulsed Electric Fields. Journal of Food Science, 68(6): 1995-2001.
15. Misra S, Ghatak S, Patil N, Dandawate P, Ambike V, Adsule S, Unni D, Swamy KV, Padhye S,2013. Novel dual cyclooxygenase and lipoxygenase inhibitors targeting hyaluronan–CD44v6 pathway and inducing cytotoxicity in colon cancer cells. Bioorganic and Medicinal Chemistry, 21: 2551–2559.
16. Moreno JJ, 2009. New aspects of the role of hydroxyeicosatetraenoic acids in cell growth and cancer development. Biochemical Pharmacology. 2009; 77: 1-10.
17. Parker CW, 1987. Lipid mediators produced throught the lipoxygenase pathway. Annual Review of Immunulogy, 5: 65-84.
18. Pérez-Gilabert M, Sánchez-Felipe I, García-Carmona F, 2005. Purification and partial characterization of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps. J Agric Food Chem., 53(9):3666-71.
19. Poeckel, D, Funk CD, 2010. The 5-lipoxygenase/leukotriene pathway in preclinical models of cardiovascular disease. Cardiovasc Res., 86, 243-253.
20. Rodrigo D, Jolie R, Van Loey A, Hendrickx M, 2007. Thermal and high pressure stability of tomato lipoxygenase and hydroperoxide lyase. Journal of Food Engineering, 79: 423-429.
21. Sacan O, Turhan Y. E,2014. Lipoxygenase Inhibitory Activities of Some Plant Extracts and Chemical Compounds. IUFS Journal of Biology, 73(2): 47-52
22. Samuelsson B, Dahlén SE, Lindgren JA, Rouzer CA, Serhan CN, 1987. Leukotrienes and lipoxins: structures, biosynthesis, and biological effects. Science, 237: 1171-1176.
23. Sarı O, 2006. Bezelye (Pısum Satıvum)’den Lipoksijenaz Enziminin Saflaştırılması Çukurova Üniversitesi Fen Bilimleri Enstitüsü, Yüksek LisansTezi.
24. Yashaswini PS, Rao AG, Singh SA, 2017. Inhibition of lipoxygenase by sesamol corroborates its potential anti-inflammatory activity. Int J Biol Macromol., 94(Pt B):781-787.
25. Yamamoto S, Suzuki H, Veda N, 1997. Arachidonate 12-lipoxygenases. Progress in Lipid Research, 36: 23-41.