Investigation of the effect of DT-15-Crown-5 crown ether on PON1 enzyme

Öz

Human serum paraoxonase 1 (PON1, EC3.1.8.1) is an important liver enzyme which is bound to HDL, contains calcium in its active site, hydrolyses organophosphate agents and nerve gases, has protective effect against the formation of lipid peroxides by oxidation of LDL and bacterial endotoxins. In our study, in order to purify the enzyme PON1, it was purified by hydrophobic interaction chromatography using sepharose-4B-L-tyrosine-1-naphthylamine gel. The purity of the enzyme was also checked by SDS - PAGE. 7,13-Dithio-1,4,10-trioxacyclopentadecane (DT-15-Crown-5) thio crown ether was synthesized as a result of the ring closure reaction according to the SN2 mechanism to be used as an inhibiting agent. Strong inhibition effect of DT-15-Crown-5 on paraoxanase enzyme was observed. Inhibition value (IC50) of DT-Taçeter with PON1 was determined as 22.62 M.

Anahtar Kelimeler

• Thiocrown ether,paraoxanase (PON),inhibition,hydrophobic interaction chromatography

PON1 enzimi üzerine DT-15-Crown-5 taç eterinin etkisinin araştırılması

Öz

İnsan serum paraoksonaz 1 (PON1, EC3.1.8.1) HDL'ye bağlı olan, aktif bölgesinde kalsiyum içeren, organofosfat ajanlarını ve sinir gazlarını hidroliz eden, LDL'nin oksidasyonu ile lipit peroksitlerin oluşumuna ve bakteri endotoksinlerine karşı koruyucu etkisi olan önemli bir karaciğer enzimidir. Çalışmamızda, PON1 enzimi daha önceden sentezi yapılmış, sepharose-4B-L-tirosin-1-naftilamin jeli kullanılarak hidrofobik etkileşim kromatografisi ile tamamen saflaştırılmıştır. Enzimin saflık kontrolü de SDS – PAGE ile yapılmıştır. İnhibisyon ajanı olarak kullanılmak üzere 7,13-dithio-1,4,10-trioksasiklopentadekan (DT-15-Crown-5) tiyo taç eteri SN2 mekanizmasına göre halka kapama reaksiyonu sonucunda sentezlendi. DT-15-Crown-5’in paraoksanaz enzimi üzerinde kuvvetli inhibisyon etkisi gözlendi. DT-Taçeterin PON1 ile inhibisyon değeri (IC50) 22,62 M olarak tespit edildi.

Anahtar Kelimeler

• Tiyo taç eter,paraoksanaz (PON),inhibisyon,hidrofobik etkileşim kromatografisi

Kaynakça

1. Broomfield, C.A., Ford, K.W., Hydrolyses of nerve gases by plasma enzymes, Proceedings of the 3rd International Meeting on Cholinesterases, La Grande-Motte, 161, 65, (1991).
2. Davies, H.C., Richter, R.J., Keifer, M., Broomfield, C.A., Sowalla, J., Furter, C.I., The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin, Nature Genetics, 14, 334, (1996).
3. Jakubowski, H., Calcium-dependent human serum homocysteine thiolactone hdyrolase. A protective mechanism against protein N-homocysteinylation, J. Biol. Chem., 275, 3957, (2000).
4. Kitchen, B.J., Masters, C.J. and Winzor, D.J., Effects of lipid removal on the molecular size and kinetic properties of bovine plasma arylesterase, Biochem J, 135, 93, (1973).
5. Mazur, A., An enzyme in animal tissue capable of hydrolyzing the phosphorus-fluorine bond of alkyl fluorophosphates, J. Biol. Chem., 164, 271-289, (1946)
6. Aldridge, W.N., Serum esterases I: two types of esterases (A and B) hydrolysing p-nitrophenyl acetate propionate and butyrate and a method their determination, Biochem J., 53,110-117, (1953).
7. Aldridge, W.N., Serum esterases II: an enzyme hyrolysing diethyl p-nitrophenyl phosphate (E600) and its identitiy with the A-esterase of mamalian sera, Biochem. J., 53, 117-124, (1953)
8. Gan, K.N., Smolen, A., Eckerson, H.W., La Du, B.N., Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities, Drug Metab. Dispos., 19, 100-106, (1991).

9. Sorenson RC., Primo-Parmo SL., Kuo CL., Adkins S., Lockridge O. and La Du BN., Reconsideration of the catalytic center and mechanism of mammalian paraoxsonase/arylesterase, Proc. Natl. Acad. Sci. USA, 92, 7187-7191, (1995).
10. Sorenson RC., Aviram, M., Bisgaier, C.L., Billecke, S., Hsu, C., La Du, B.N., Properties of retained N-terminal hydrophobic leader sequence in human serum paraoxonase/arylesterase, Chem. Biol. Interact., 119-120, 243-249, (1999).
11. Main, A.R., The role of A- esterase in the acute toxicity of paraoxon, TEPP and parathion, Can. J. Biochem. Physiol., 34, 197-216, (1956).
12. Aviram, M., Hardak, E., Vaya, J., Mahmood, S., Milo, S., Hoffman, A., Billecke, S., Draganov, D. and Rosenblat, M., Human serum paraoxonases (PON1) Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities, Circulation, 101, 2510, (2000).
13. Azarsız, E. and Sözmen, E.Y., Paraoksonaz ve Klinik Önemi, Türk Biyokimya Dergisi, (2000)
14. La Du, B.N., Pharmagenetics of drug Metabolism, Pergamon, Elmsford, NY, 51-91, (1992)
15. Rodrigo, L., Gil, f., Hernandez,A.F., Pla, a., Identification of two rat liver proteins with paraoxonase activity: biochemical evidence for the identitiy of paraoxonase and arylesterase, Chem. Biol. Interact. 119-120,263-275, (1999).
16. Sozmen, B., Delen, Y., Girgin, F.K., Sözmen, E.Y., Catalase and paraoxonase in hypertensive type 2 diabetes mellitus: correlation with glycemic control, Clinical Biochemistry, 32,423-427, (1999).
17. Rolls, Bj, Effects of Intense Sweeteners on Hunger, Food-Intake, and Body-Weıght, Amerıcan Journal of Clınıcal Nutrıtıon, 53, 872-878, (1991).
18. Zarate, F.M., Schulwitz, S.E., Stevens, K.J., Venables, B.J., Bioconcentration of triclosan, methyl-triclosan, and triclocarban in the plants and sediments of a constructed wetland source, Chemosphere, 88, 323-329, (2012).
19. Billecke, S., Draganov, D., Counsell, R., et al., Human serum paraoxonase (PON1) isozymes Q and R hydrolyze lactones and cyclic carbonate esters, Drug Metab Dispos, 28, 35-41, (2000).
20. Harel, M., Aharoni, A., Gaidukov, L., et al., Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes, Nat Struct Mol Biol., 11, 412–419, (2004).
21. Kockar, F., Sinan, S., Yildirim, H., Arslan, O., Differential effects of some antibiotics on paraoxonase enzyme activity on human hepatoma cells (Hep62) in vitro, J. Enzyme Inhib Med Chem, 25, 715-719, (2010).
22. Erzengin, M., Basaran, I., Cakir, U., et al., In vitro inhibition effects of some dihydroxycoumarin compounds on purified human serum paraoxonase I (PON1), Appl Biochem Biotechnol, 168, 1540–1548, (2012).
23. Senturk, M., Ekinci, D., Alici, H.A., Beydemir, S., Paraoxonase-1, an organophosphate detoxifier and cardioprotective enzyme is inhibited by anasthetics: an in vitro and in vivo insight, Pest Biochem Physiol, 11, 206-211, (2011).
24. Turkes C, Soyut H, Beydemir S. Effect of calcium channel blockers on paraoxonase-1 (PON1) activity and oxidative stress, Pharmacol Rep, 66,74-80, (2014).
25. Pla, A., Rodrigo, L., Hernandez, A.F., et al., Effect of metal ions and calcium on purified PON1 and PON3 from rat liver, Chem Biol Interact, 167, 63-70, (2007).
26. Gencer, N., Arslan, O., In vitro effects of some pesticides on PON1Q192 and PON1R192 isoenzymes from human serum, Fresen Environ Bull, 20, 590-596, (2011).
27. Black, D.St.C., McLean, I.A., Tetrahedron Lett., 39, 61, (1969).
28. Izatt, R.M., Pawlak, K., Bradshaw, J.S., Bruening, R.L., Chem. Rev., 91, 1721, (1991).
29. Craig, A.S.. Kataky, R., Matthews, R.C., Parker, D., Ferguson, G., Lough, A., Adams, H., Bailey, N., Schneider, H., J. Chem.Soc., 2, 1523, (1990).
30. Bradshaw, J.S., Izatt, R.M., Acc. Chem. Res., 30, 338, (1997).
31. Krylova, K., Kulatilleke, C.P., Hegg, M.J., Salhi, C.A.,. Ochrymowycz, L.A, Rorabacher, D.B., Inorg. Chem., 38, 4322, (1999).
32. van de Water, L.G.A., Driessen, W.L., Glenny, M.W., Reedijk, J., Schröder M., React. Funct. Polym., 51, 33, (2002).
33. Çiçek, B., Yildiz, A., Synthesis Metal Ion Complexation and Computational Studies of Thio Oxocrown Ethers, Molecules, 16, 8670-83, (2011).
34. Sinan, S., Kockar, F., Arslan, O., Novel purification strategy for human PON1 and inhibition of the activity by cephalosporin and aminoglikozide derived antibiotics, Biochimie, 88, 565-574, (2006).
35. Karataş, M.O., Uslu, H., Alıcı, B., Gökçe, B., Gencer, N., Arslan, O., Some coumarins and benzoxazinones as potent paraoxonase 1 inhibitors, J Enzyme Inhib Med Chem., 31, 1386-1391, (2016).
36. Senturk, M., Ekinc,i D., Alici, H.A., Beydemir, S., Paraoxonase-1, an organophosphate detoxifier and cardioprotective enzyme is inhibited by anasthetics: an in vitro and in vivo insight, Pest Biochem Physiol, 11, 206-211, (2011).
37. Turkes, C., Soyut, H., Beydemir, S., Effect of calcium channel blockers on paraoxonase-1 (PON1) activity and oxidative stress, Pharmacol Rep 66, 74-80, (2014).
38. Pla, A., Rodrigo, L., Hernandez, A.F., et al., Effect of metal ions and calcium on purified PON1 and PON3 from rat liver, Chem Biol Interact, 167, 63-70, (2007).
39. Gencer, N., Arslan, O., In vitro effects of some pesticides on PON1Q192 and PON1R192 isoenzymes from human serum, Fresen Environ Bull, 20, 590-596, (2011).
40. Ergün, A., Çiçek, B., Bazı makrohalkalı tiyocrown eterlerin polifenol oksidaz enzimi üzerindeki inhibisyon etkilerinin araştırılması, Balıkesir Üniversitesi Fen Bilim Enstitüsü Dergisi, 20, 1-8, (2018).
41. Akkemik, E., Çalışır, Ü., Çiçek, B., İnsan karbonik anhidraz I,II izoenzim aktiviteleri üzerine bazı tiyocrown eterlerin etkisi, Balıkesir Üniversitesi Fen Bilim Enstitüsü Dergisi,;19, 192-199, 2017.
42. Çiçek, B., Ergün, A., Gencer, N., Synthesis and Evaluation in vitro Effects of Some Macrocyclic Thiacrown Ethers on Erythrocyte Carbonic Anhydrase I and II, Asian Journal of Chemistry, 24, 3729-3731, (2012).
43. Akkemik, E., Çiçek, B., Camadan, Y., Çalışır, U., Onbasioglu, Z., The determination of the carbonic anhydrases activators in vitro effect of mixed donor crown ethers, Journal of Biochemical and Molecular Toxicology, 32, 3, (2018).