Yeni Sentezlenmiş Katyonik Stiril Boyanın Biyomoleküllerle Etkileşiminin Spektroskopik ve Moleküler Kenetlenme Metotları ile İncelenmesi

Abstract

In this thesis, a novel cationic styryl dye given as (E)-2-(4-(dibütilamino)stiril)-1,3,3-trimetil-3H-indol-1-yum iyodür (Ci5) was synthesized and its spectral properties were characterized. The interactions of the Ci5 compound with the protease enzymes trypsin at pH 7.4 and pepsin at pH 2.0 in buffer solutions were studied by multiple spectroscopic (fluorescence, absorption, FTIR and CD) and molecular docking methods. In addition, the interaction between Ci5-DNA was analyzed in a spectroscopic methods and groove binding was observed. In fluorimetric studies, it was concluded from the data obtained from fluorescence quenching that the interaction of pepsin/trypsin with the Ci5 compound occurs by static quenching mechanism. It has shown that the interaction between the Ci5 compound and the enzymes is based on complex formation. The type of binding and its properties are described based on the thermodynamic parameters (H ve S) obtained at different temperatures. The positive S and negative H parameters indicated that both electrostatic forces and hydrophobic interactions also have a role in the interaction of Ci5 and pepsin. Negative ΔS and negative ΔH values indicated the presence of van der Waals forces and hydrogen bonds in the interaction of trypsin with Ci5. The shifts in the amide bands of pepsin/trypsin in FTIR studies indicated the presence of interaction between the dye and the enzyme. And also CD results indicated that the binding of Ci5 to pepsin/trypsin caused to secondary structure changes of enzymes, with the loss of helical stability.

According to Forster theory, the distance (r) between donor-acceptor molecular pairs was found as 3.53 nm in the Ci5-pepsin pair and 3.27 nm in the Ci5-tripsin pair. It was concluded that resonance energy transfer between dye/enzyme pairs occurs in a non-radiation way.The obtained experimental data were supported and validated by molecular docking results. In view of on the molecular docking data, it was concluded that the pepsin/trypsin of Ci5 binds to certain amino acid residues and to the nucleotides of DNA. In addition, binding energy and bond distances were calculated.

The analytical data obtained for the determination of Ci5 in the presence of pepsin and trypsin, the limit of dedection (LOD) is 1.324x10-5 M in the presence of pepsin and 4.238x10-6 M in the presence of trypsin. The limit of quantification (LOQ) was calculated as 4.414x10-5 M in the presence of pepsin, 1.413x10-5 M in the presence of trypsin.