Hasan KARAGEÇİLİ, Mustafa Oğuzhan KAYA, Emrah YERLİKAYA, Ramazan DEMİRDAĞ

Siirt Tiftik Keçisi Böbrek Dokusundan Karbonik Anhidraz Enziminin Saflaştırılması, Karakterizasyonu ve Bazı Biyokimyasal Özelliklerinin Araştırılması

Karbonik anhidrazlar (CA) (E.C:4.2.1.1) bakteri gibi en basit organizmalardan bitki ve hayvan gibi en kompleks canlılara kadar yaygın şekilde dağılmış çinko içeren bir grup metaloenzimdir. CA inhibitörleri köpek ve kedilerin glukoma tedavisinde kullanılan başlıca ilaçların birkaçıdır. CA enzimleri daha önceki çalışmalarda afinite kromatografisi yöntemi ile başarılı bir şekilde koyun, tavuk, balık, sığır ve insan gibi pek çok canlıdan saflaştırılmış ve karakterize edilmiştir. Bu çalışmada ise CA enzimi, Siirt Tiftik Keçisi böbrek dokusundan Sepharose-4B-L-tirozin-sülfanilamid afinite kolonu kullanılarak 902,9 EU x mg-1 spesifik aktivite ile % 50,19 verimle 83,54 kat saflaştırıldı. Saflaştırılan enzimin saflığı SDS-PAGE ile doğrulandı. Siirt Tiftik Keçisi böbrek CA enziminin karakterizasyonu yapıldığında; optimum iyonik şiddeti=25 mM, optimum sıcaklığı=45ºC, optimum pH'sı=8,5 ve stabil pH'sı=7,0 olarak belirlendi. Saflaştırılan CA enzimi üzerine Al3+, Ni2+, Cd2+, Pb2+, Ba2+, Zn2+, B3+, Fe3+, Se2+, Ag+ ve Co2+ metal iyonlarının inhibitör etkileri incelendi. İnhibisyon gösteren metallerin IC50değerlerini bulmak amacıyla inhibisyon grafikleri çizildi.

Purification and Characterization of Carbonic Anyhdrase Enzyme from The Siirt Mohair Goat Kidney Tissue and Investigation of Some Biochemical Properties

Carbonic anhydrase (CA) (E.C:4.2.1.1) is a group of metalloenzyme containing zinc widely distributed from the simplest organisms such as bacteria to the most complex organisms such as plants and animals. CA inhibitors are some of the principal drugs used in the management of canine and feline glaucoma. In earlier studies; CA enzymes successfully have been purified and characterized from many living things such as; sheep, chicken, fish, bovine and human. In this study, the CA enzyme has purified from Siirt Mohair Goat kidney tissue with 902.9 EU x mg-1 of specific activity, 50.19% of purification yield and 83.54 of purification folds by using Sepharose-4B-L-tyrosine-sulfonamide affinity column. The purity of the purified enzyme has confirmed by SDS-PAGE. As the characterization of CA enzyme's in Siirt Mohair Goat kidney has been done; the optimum ionic strength=25mM, the optimum pH=8.5, the optimum temperature=45ºC and the stable pH=7.0 has been determined. Inhibitory effects of Al3+, Ni2+, Cd2+, Pb2+, Ba2+, Zn2+, B3+, Fe3+, Se2+, Ag+ and Co2+metal ions have been examined on the purified CA enzyme. Inhibition graphics have been drawn in order to find the IC50 values of metals showing inhibition.

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Berg J, Tymoczko J, Stryer L 2002. Biochemistry, 5th Ed., W.H. Freeman Co.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248.
Bülbül, M., Saraçoğlu, N., Küfrevioğlu, Ö.İ., Çiftçi M. 2002. Bile acid derivatives of 5-amino-1.3,4-thiadiazole-2 sulfonamide as new carbonic anhydrase inhibitors: Syntesis and investigation of inhibition effects. Bioorganic and Medicinal Chemistry, 10, 2561.
Chegwidden, W.R., Edwards, Y., Carter, N. 2000. The Carbonic Anhydrases-New Horizons. Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly,W. S., and Valle, D., eds) 8th Ed.,2165-2204, McGraw-Hill, Inc., NewYork.
Christensen GM, Tucker JH. 1976. Effects of selected water toxicants on the in vitro activity of fish carbonic anhydrase. Chem Biol Interact, 13, 181-192.
Çankaya, M., Kuzucu, M. 2015. Effect of Some Drugs on Sheep Liver Carbonic Anhydrase I and II. Erzincan University Journal of Science and Technology, 8-2, 201-212.
Demirdağ R., Yerlikaya E., Küfrevioğlu Ö.İ. (2012). Purification of carbonic anhydrase-II from sheep liver and inhibitory effects of some heavy metals on enzyme activity. Journal of Enzyme Inhibition and Medicinal Chemistry, 27-6, 795-799.
Demirdağ R., Yerlikaya E., Şentürk M., Küfrevioğlu Ö.İ., Supuran C.T. 2013. Heavy metal ion inhibition studies of human, sheep and fish ?-carbonic anhydrases. Journal of Enzyme Inhibition and Medicinal Chemistry, 28-2, 278-282.
Doğanay S.,Fırat P.G. 2007. Karbonik Anhidraz İnhibitörleri. Journal of Glaucoma-Cataract, 2, 213-218.
Ekinci D, Beydemir S, Küfrevioglu OI. In vitro inhibitory effects of some heavy metals on human erythrocyte carbonic anhydrases. Journal of Enzyme Inhibition and Medicinal Chemistry, 2007,22,745-750.
Iqbal S., Rahman N., Iqbal J. 2014. A capillary electrophoresis-based enzyme assay for kinetics and inhibition studies of carbonic anhydrase.Anal Biochem, 444, 16-21.
Kaya E.D., Söyüt H., Beydemir Ş. 2015. The toxicological impacts of some heavy metals on carbonic anhydrase from gilthead sea bream (Sparus aurata) gills. Environmental Toxicology and Pharmacology. 39,825-832
Laemmli, D.K., 1970. Cleavage of structural proteins during in assembly of the head of Bacteriophage T4. Nature, 227, 680.
Lineweaver, H., and Burk, D. 1934. The determination of enzyme dissocation constants. J. Am. Chem. Soc, 57, 685.
Maslanka, T. 2015. A review of the pharmacology of carbonic anhydrase inhibitors for the treatment of glaucoma in dogs and cats. Veterinary Journal. 203, 3, 278-284.
Söyüt H., Beydemir Ş. 2012. The impact of heavy metals on the activity of carbonic anhydrase from rainbow trout (Oncorhynchus mykiss) kidney. Toxicology and Industrial Health, 28, 4, 296-305.
Supuran, C.T. and Scozzafava, A., 2001. Carbonic Anhydrase Inhibitors. Current Medicinal Chemistry, 1, 61-97.
Supuran, C.T., Scozzafava, A. 2000. Carbonic anhydrase inhibitors -Part 94. 1,3,4-Thiadiazole-2-sulfonamide derivatives as antitumor agents? European Journal of Medicinal Chemistry, 35, 9, 867-874.
Verpoorte, J.A., Mehta, S., and Edsall, J.T. 1967. Esterase activities of human carbonic anhydrase. The Journal of Biological Chemistry, 242, 4221.
Yerlikaya E. 2012. İnsan Karbonik Anhidraz IX İzoenziminin Klonlanması, Üretilmesi, Antitümör İlaçların ve Bazı Kimyasalların Enzim Aktivitesi Üzerine Etkilerinin İncelenmesi, Doktora Tezi, Erzurum.