Gümüşhane Harşit Çayı'ndaki Gökkuşağı Alabalığı (Oncorhynchus mykiss) solungacından karbonik anhidraz enziminin saflaştırılması ve bazı ağır metallerin enzim aktivitesi üzerine inhibisyon etkilerinin incelenmesi

Öz

Bu çalışmada Gökkuşağı alabalığı (Oncorhynchus mykiss) solungacından karbonik anhidraz (karbonat hidroliyaz E.C.4.2.1.1) enzimi saflaştırılmış ve bazı ağır metallerin enzim aktivitesi üzerine inhibisyon etkileri incelenmiştir. Saflaştırma işlemi, homojenat hazırlandıktan sonra bu homojenatın afinite kolonuna yüklenerek elüsyon yapılması yoluyla gerçekleştirilmiştir. Gökkuşağı alabalığı (Oncorhynchus mykiss) solungacından karbonik anhidraz (CA) enziminin saflaştırılmasında %25,1 verim elde edilmiştir. Enzimin saflığı SDS poliakrilamid jel elektroforezi ile tek bant elde edilerek tespit edilmiştir. Ayrıca bazı ağır metallerin CA enzimi aktivitesi üzerine inhibisyon etkileri incelenmiştir. Bu amaçla Al, Sr, Ca, Hg, Mn, Fe, Ba, Mg, Zn ve Cu metal inhibitörler olarak kullanıldı. Tüm metaller, CA enzimi üzerinde inhibisyon etki göstermiştir. Metal iyonlarının IC50 değerleri, % aktivite ile inhibitör konsantrasyonu [I] arasında çizilen grafikle belirlendi. Al, Sr, Ca, Hg, Mn, Fe, Ba, Mg, Zn ve Cu’ın IC50 değerleri sırasıyla 0,0047, 0,015, 0,039, 0,0097, 0,022, 0,012, 0,0085, 0,02, 0,0063 ve 0,011 mM olarak tespit edildi.

Anahtar Kelimeler

• Ağır metal
• Gökkuşağı alablalığı
• Harşit çayı
• karbonik anhidraz

Destekleyen Kurum

Bu çalışma, Gümüşhane Üniversitesi Bilimsel Araştırma Projeleri Birimi tarafından 14.A0114.02.01 numaralı proje kapsamında (GÜBAP2902: Hızlı Destek Programı) desteklenmiştir. Tüm deneysel çalışmalar, Ağrı İbrahim Çeçen Üniversitesi Merkezi Araştırma Laboratuvarı’nda gerçekleştirilmiştir.

Proje Numarası

14.A0114.02.01

Purification of carbonic anhydrase from the gill tissue of rainbow trout (Oncorhynchus mykiss) inhabiting the Harşit Stream (Gümüşhane, Türkiye) and investigation of the inhibitory effects of some heavy metals on enzyme activity

Öz

In this study, carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) was purified from the gill tissue of rainbow trout (Oncorhynchus mykiss), and the inhibitory effects of several heavy metals on enzyme activity were investigated. The purification procedure involved preparing a gill homogenate, followed by affinity chromatography and subsequent elution of the enzyme. Carbonic anhydrase (CA) was purified from the gill tissue of Oncorhynchus mykiss with a purification yield of 25.1%. The purity of the enzyme was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), which revealed a single protein band. The inhibitory effects of several metal ions on CA activity were also evaluated. For this purpose, Al, Sr, Ca, Hg, Mn, Fe, Ba, Mg, Zn, and Cu were used as inhibitors. All tested metal ions exhibited inhibitory effects on carbonic anhydrase activity. The IC₅₀ values of the metal ions were determined from plots of residual enzyme activity (%) versus inhibitor concentration ([I]). The IC₅₀ values for Al, Sr, Ca, Hg, Mn, Fe, Ba, Mg, Zn, and Cu were determined to be 0.0047, 0.015, 0.039, 0.0097, 0.022, 0.012, 0.0085, 0.020, 0.0063, and 0.011 mM, respectively.

Anahtar Kelimeler

• Heavy metal
• raimbow trout
• harşit stream
• carbonic anhydrase

Destekleyen Kurum

This study was financially supported by the Scientific Research Projects Unit of Gümüşhane University under Project No. 14.A0114.02.01 (GÜBAP2902: Rapid Support Program). All experimental procedures were conducted at the Central Research Laboratory of Ağrı İbrahim Çeçen University.

Proje Numarası

14.A0114.02.01

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