calcoaceticus strain Pediococcus acidilactici Örnek No (mm)
Formül Çap(mm) Çap(mm)
0 Kontrol 1 C15H13N(113) 22 23 2 C15H12BrN(116) 22 22 3 C16H15NO(163) 18 20 4 C16H14FNO(164) 16 17 5 C16H14ClNO(166) 23 22 6 C16H14ClNO(167) 15 19 7 C16H14BrNO(169) 22 24 8 C16H14BrNO(170) 15 21
hCA-I ve hCA-II izoenzimleri insan kanından Sepharose 4B-L tyrosine- sulphanylamide afinite kromatografisi kullanılarak tek basamakta saflaştırıldı. hCA-I 797.33 spesifik aktivite ile 2953.1 kat ve % 60.2 verimle saflaştırıldı, hCA-II ise 797.33 spesifik aktivite ile 7497.2 kat ve % 52.3 verimle saflaştırıldı (Çizelge 5.2). Yeni sentezlenen sinnamaldehit-anilin ve Parametoksisinnamaldehit-anilin türevlerinin hCA-I ve hCA-II izoenzimlerinin aktivitesi üzerine inhibitör etkileri in vitro olarak test edildi, I50
değerleri tüm yeni sentezlenen sinnamaldehit-anilin ve Parametoksisinnamaldehit-anilin türevleri için çizilen % Aktivite-[İnhibitör] grafiklerinden hesaplandı (Şekil 5.4 (Fig.1-8)) ve çizelge 5.3'te verildi. Karbonik anhidraz enzimi çok sayıda farklı organizmadan pek çok kez saflaştırıldı ve enzim aktiviteleri üzerine çeşitli pestisid, kimyasal ve ilaçların etkisi araştırılmıştır. Bu çalışmada, hCA-I ve hCA-II izoenzimleri Sepharose 4B-L-tirosin- sulfanilamid afinite kromatografisi ile saflaştırıldı ve CO2'i substrat olarak kullanılarak
65
hidrataz aktivite yöntemi ile yeni sentezlenen sinnamaldehit-anilin ve Parametoksisinnamaldehit-anilin türevleri için kinetik çalışmalar yapıldı. Sonuçlar Çizelge 5.2’te verildi.
Çizelge 5.2. İnsan eritrositinden hCA I ve hCA II'nin saflaştırılmasının özeti.
Saflaştırma Adımları Haci m Aktivite Toplam aktivite Protein Özel Aktivite Saflaştırma Katsayısı Ml (EÜ/mL) EÜ % (mg/mL) (EÜ/mg)
Hemolysate 100 44.67 4467 100 1.64*103 0,27 -
hCA-I 45 59.8 2691 60.2 0.075 797,33 2953.1
hCA-II 35 66.8 2338 52.3 0.033 2024,24 7497.2
Çizelge 5.3. Yeni sentezlenen sinnamaldehit-anilin ve parametoksi
sinnamaldehit-anilin türevleriyle hCA- I ve hCA-II'nin in vitro inhibisyonu için I50 değerleri. Kimyasallar CA-I (mM) I50 CA-II (mM) I50 113 C15H13N - - 116 C15H12NBr 1,24 1,844 163 C16H15NO 1.55 1.04 164 C16H14FNO 1,42 2,6 166 C16H14NOCl 0.99 0,843 167 C16H14NOCI 1,89 1,85 169 C16H14NOBr 1,22 2,74 170 C16H14NOBr 1,94 1,91 Acetazolamide (AAZ) 5.2 6.1
66
Elde edilen bulgulardan tüm Schiff bazlarının asetazolamid referans bileşiğine karşı inhibitör etkinliğinin daha fazla olduğu belirlendi. hCA-I ve hCA-II izoenzimleri üzerine en fazla etkiyi C16H14NOCl (166) bileşiğinin sırasıyla 0.99 M ve 0.843 M IC50
değerlerine sahiptir. Elde edilen bulgulardan yeni sentezlenen schiff bazlarının hCA-I ve hCA-II izoenzimlerinin üzerine güçlü inhibisyon etkisi gösterdikleri ve ileriki çalışmalara yeni CA enzimi inhibitörlerinin oluşturulmasında temel oluşturacağı düşünülmektedir (Altintop et al. 2015; Altintop et al. 2017; Türk et al. 2017).
Şekil 5.4 (Fig.1-8). İnsan eritrositlerinden saflaştırılan CA-I ve CA-II enzim aktiviteleri üzerine ilaçların in vitro etkileri
y = 7.2251x + 82.284 R² = 0.9658 y = 42.194x + 92.853 R² = 0.9775 0 50 100 150 200 250 0 1 2 3 4 % A k ti v ite [C15H13N], M CA-I CA-II y = 121.9e-0.724x R² = 0.9636 y = 102.27e-0.388x R² = 0.9676 0 20 40 60 80 100 120 0 1 2 3 4 % A k ti v ite [C15H12NBr], M CA-I CA-II
67 y = 110.28e-0.51x R² = 0.9776 y = 102.48e-0.718x R² = 0.987 0 20 40 60 80 100 120 0 0.5 1 1.5 2 2.5 3 3.5 % B ağı l ak ti vi te [C16H15NO ], M CA-I CA-II y = 103.05e-0.51x R² = 0.9881 y = -17.041x + 94.375 R² = 0.9823 0 20 40 60 80 100 0 1 2 3 4 % B ağı l A k ti vi te [C16H14FNO], M CA-I CA-II
68 y = 102.48e-0.718x R² = 0.987 y = 93.918e-0.748x R² = 0.9783 0 20 40 60 80 100 120 0 0.5 1 1.5 2 2.5 3 3.5 B ağı l ak ti vi te ,(%) [C16H14NCl ], M CA-I CA-II y = -19.561x + 86.962 R² = 0.9967 y = -23.095x + 90.407 R² = 0.9848 0 20 40 60 80 100 0 1 2 3 4 % B ağı l A k ti vi te [C16H14NOCl], M CA-I CA-II Linear (CA-II)
69
İmin bileşiklerinin çeşitli kompleksleri üzerinde spektroskopik olarak çalışılabilir ve kristal yapısındaki değişimler incelenebilir. İmin türevlerinden yeni organometalik bileşiklerin sentezinde, metal iyonlarının ve sensörlerin hazırlanmasında kullanılabilir. Bu iminlerin fotokromik veya termokromik özellikleri araştırılabilir. İminler farklı yöntemlerle aminlere indirgenip, farklı alanlarda kullanılabilir.
Bu tez kapsamında elde edilen sonuçlar doğrultusunda çalışmanın bir kısmını içeren sonuçlar ile hazırlanan yayın Fresenius Environmental Bulletin dergisinde yayımlanmıştır (Celik et al. 2018).
y = 95.601e-0.532x R² = 0.9785 y = -15.233x + 91.763 R² = 0.9919 0 20 40 60 80 100 0 1 2 3 4 % B ağı l A k ti vi te [C16H14NOBr], mM CA-I CA-II y = -18.032x + 84.899 R² = 0.9931 y = -20.563x + 89.252 R² = 0.9965 0 20 40 60 80 100 0 0.5 1 1.5 2 2.5 3 3.5 4 % B ağı l A k ti vi te [C16H14NOBr], M CA-I CA-II
70
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