Zuhal ALIM, Bedia ÇAMUR, Şükrü BEYDEMİR, Ömer İrfan KÜFREVİOĞLU

Orkinos Thunnus Thynnus Linnaeus, 1758 solungacında karbonik anhidraz enzim aktivitesi ve bazı metal derişimleri arasındaki ilişki

B ugün teknoloji ve sanayideki gelişmelerle birlikte metal kirliliği hızla artmaktadır. Ağır metaller biyobirikimleri ve doğada çözülmeyen özelliklerinden dolayı tüm dünyada sucul organizma için önemli kirliliktir. Metal toksisitesi, metabolizmada DNA hasarı, lipid peroksidasyonunun artması, protein sulfidril gruplarının oksidasyonu ve enzim inaktivasyonu gibi oksidatif hasara neden olur. Bu çalışmada, balık metabolizmasında hayati rol oynayan karbonik anhidraz enzimi üzerine bazı metallerin in vitro etkilerini inceledik. Bu amaçla enzim orkinos solungaçlarından Sepharose 4B–anilin–sulfanilamid afinite kromatografisi yöntemi kullanılarak 1062 EÜ/mg protein spesifik aktivite ve %31 verimle saflaştırıldı. SDS-PAGE jel elektroforezinde yaklaşık 29 kDa’da tek bant görüldü. İn vitro koşullar altında hidrataz yöntemi kullanılarak metallerin Ag+, Cu2+, Pb2+, Zn2+, Cd2+, Co2+ farklı derişimlerinde inhibisyon etkileri belirlendi. İn vitro inhibisyon derecesi Ag+> Cu2+> Pb2+> Zn2+ > Cd2+> Co2+ olarak belirlendi. Bu sonuçlardan Ag+’nin güçlü bir CA enzim inhibitörü olduğu görüldü

Anahtar Kelimeler:

Karbonik anhidraz, metal toksisitesi, inhibisyon

The correlation between some metal concentrations and carbonic anhydrase activity in Tuna Thunnus Thynnus Linnaeus, 1758 Gill

Today metal pollution is rapidly increasing with advances in technology and industry. Due to their bioaccumulative and nonbiodegradable properties, heavy metals are important contaminant of aquatic organism all over the world. Metal toxicity causes oxidative damage such as DNA damage, enhanced lipid peroxidation, the oxidation of protein sulfydryl groups and enzyme inactivation in the metabolism. In this study, we investigated in vitro effects of some metals on carbonic anhydrase enzyme which has vital role in fish metabolism. For this aim, The enzyme, was purified from gills of tuna with a specific activity of 1062 EU/mg proteins and 31% yield using Sepharose 4B– aniline–sulfanilamide affinity chromatography method. SDS–polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approximately 29 kDa. Inhibitory effects of metals Ag+, Cu2+, Pb2+, Zn2+, Cd2+, Co2+ on CA activity were determined at different concentrations using the hydratase method under in vitro conditions. Consequently, in vitro inhibition rank order was determined as Ag+> Cu2+> Pb2+> Zn2+ > Cd2+> Co2+. From these results, we showed that Ag+ is the most potent inhibitor of CA enzyme.

Keywords:

Carbonic anhydrase, metal toxicity, Inhibition,

PDF

___

1. M. Sevcikova, H. Modra, A. Slaninova, Z. Svobodova, Metals as a cause of oxidative stress in fish, Veterinarni Medicina, 56 (2011) 537–546.
2. H. Soyut, S. Beydemir, The impact of heavy metals on the activity of carbonic anhydrase from rainbow trout (Oncorhynchus mykiss) kidney, Toxicol Ind Health, (2011), DOI:10.1177/0748233711410914.
3. D. Ekinci, S. Beydemir, O.I. Küfrevioglu, In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases, Enzyme Inhib Med Chem, 22 (2007) 745–750.
4. S.B. Ceyhun, M. Sentürk, E. Yerlikaya, O. Erdogan, O.I. Küfrevioglu, D. Ekinci, Purification and characterization of carbonic anhydrase from the teleost fish Dicentrarchus labrax (European seabass) liver and toxicological effects of metals on enzyme activity, Environ Toxicol Pharmacol, 32 (2011) 69–74.
5. C.T. Supuran, A. Scozzafava, Carbonic anhydrases as targets for medicinal chemistry. Bioorg Med Chem, 15 (2007) 4336–4350.
6. C.T. Supuran, Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators, Nat Rev Drug Discov, 7 (2008) 168–81.
7. O. Hisar, S. Beydemir, M. Bulbul, T. Yanık, Kinetic properties of carbonic anhydrase purified from gills of rainbow trout (Oncorhynchus mykiss), J. Appl. Anim. Res. 30 (2006) 185–187.
8. S. Sender, K. Böttcher, Y. Çetin, G. Gros, Carbonic Anhydrase in the Gills of Seawater and Freshwateracclimated Flounders Platichthys flesus: Purification, Characterization, and Immunohistochemical Localization, The Journal of Histochemistry & Cytochemistry, Volume 47 (1999) 43–50.
9. K.M. Gilmour, S.F. Perry, Carbonic anhydrase and acid– base regulation in fish, The Journal of Experimental Biology, 212 (2009) 1647-1661.
10. R.C. Playle,. Modelling metal interactions at fish gills, The Science of the Total Environment, 219 (1998) 147- 163.
11. S. Beydemir, I. Gulcin, O. Hisar, O.I. Kufrevioglu, T. Yanik, Effect of melatonin on glucose-6-phosphate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro and in vivo, Journal of Applied Animal Research, 28 (2005) 65–68.
12. K.M. Wilbur, N.G. Anderson, Electrometric and colorimetric determination of carbonic anhydrase, J Biol Chem,176 (1948) 147–154.
13. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding, Anal Biochem, 72 (1976) 248–254.
14. D.K. Laemmli, Cleavage of structural proteins during assembly of the head of bacteriophage T4, Nature,227 (1970) 680–683.
15. E. Raspanti, S.O. Cacciola, C. Gotor, L.C. Romero, I. Garcı´a, Implications of cysteine metabolism in the heavy metal response in Trichoderma harzianum and in three Fusarium species, Chemosphere, 76 (2009) 48–54.
16. D. Ekinci, S. Beydemir ¸ Risk assessment of pesticides and fungicides for acid–base regulation and salt transport in rainbow trout tissues, Pesticide Biochemistry and Physiology, 97 (2010) 66–70.
17. A. Farkas, J. Salanki, A. Speczıar, I. Varanka, Metal pollution as health indicator of lake ecosystems, International Journal of Occupational Medicine and Environmental Health, 14 (2001) 163-170.
18. O. Hisar, S. Beydemir, I. Gulcin, O.I. Küfrevioğlu, C.T. Supuran, Effects of low molecular weight plasma inhibitors of rainbow trout (Oncorhynchus mykiss) on human erythrocyte carbonic anhydrase-II isozyme activity in vitro and rat erythrocytes in vivo, Journal of Enzyme Inhibition and Medicinal Chemistry, 20 (2005) 35–39.
19. R.B. Voegborlo, A.M. El-Methnani, M.Z. Abedin, Mercury, cadmium and lead content of canned tuna fish, Food Chemistry, 67 (1999) 341-345.
20. M.M. Storelli, R.G. Stuffler, G.O. Marcotrigiano, Total and methylmercury residues in tuna-fish from the Mediterranean sea, Food Additives and Contaminants, 19 (2002) 715-720.
21. H. Soyut, S. Beydemir, O. Hisar, Effects of some metals on carbonic anhydrase from brains of rainbow trout, Biol Trace Elem Res, 123 (2008) 179–190.
22. H. Soyut, S. Beydemir, Purification and some kinetic properties of carbonic anhydrase from rainbow trout (Oncorhynchus mykiss) liver and metal inhibition, Protein Pept Lett, 15 (2008) 528–535.