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Study of the binding interaction of tyrosine kinase inhibitors with serum albumin
Rodová, Marie ; Indra, Radek (advisor) ; Heidingsfeld, Olga (referee)
Sunitinib and vandetanib are anti-cancer medications prescribed for medullary thyroid cancer (in the case of vandetanib) and for renal cell carcinoma, gastrointestinal stromal tumor, and pancreatic cancer (in the case of sunitinib). They belong to the group of tyrosine kinase inhibitors and act by exhibiting anti-angiogenic effects and by inhibiting tumor cell proliferation and survival through VEGFR. Additionally, vandetanib also inhibits tumor cell survival via EGFR and RET. In the presented thesis, we investigated the binding interaction between serum albumin and the TKIs vandetanib and sunitinib using BSA, HSA, and blood plasma. We examined the differences in interaction between the TKIs and various serum albumins, including pure BSA, pure HSA, and blood plasma, as well as the nature and location of the binding interaction. Additionally, we studied the influence of other ligands on this interaction and the photosensitivity of sunitinib itself. Utilizing spectroscopic techniques, including UV-VIS absorption and fluorescence quenching, we have determined the Stern-Volmer and binding constants, as well as the thermodynamic parameters, for the binding interactions of sunitinib and vandetanib with BSA and HSA. Our results indicate that complex formation occurs between BSA and sunitinib, BSA and...
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Binding of organic dyes to proteins. Applications in practical course in biochemistry.
Hynková, Anna ; Hudeček, Jiří (advisor) ; Koblihová, Jitka (referee)
In this submitted thesis the possibility of creating a new laboratory task for advanced practical courses in biochemistry, concerning the binding of a low-molecular compound (dye) to a protein macromolecule, was experimentally verified. First intention was to modify the existing task "Dialysis kinetics" with a binding of fluorescein to a protein. However, the experiments have proved, that fluorescence measurements of this kind are not reproducible in the conditions of practical courses laboratory and absorbance measurements have low sensitivity. Therefor a whole new task was created: "Binding of bromophenol blue to serum albumin", in which the stoichiometry of this binding is studied using so-called Job plot. After the optimization of procedure a laboratory task instructions were created, which are attached to this thesis. Keywords: fluorescein, bromophenol blue, serum albumin
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MRI contrast agents for angiography
Urbanovský, Peter ; Kotek, Jan (advisor) ; Vojtíšek, Pavel (referee)
Modern diagnostic method magnetic resonance imaging (MRI) usually uses contrast agents T1-type, which are based on Gd3+ complexes. Due to severe toxicity of free Gd3+ , it is desired to have thermodynamically stable and kinetically inert complexes with fast elimination from the body. This work summarizes information about a novel contrast agent based on ligand DO3AP (1,4,7,10-tetraazacyclododecane-1-methyl(alkyl)phosphinic-4,7,10- triacetic acid) with pendant hydrophobic dibenzylamino group which is able to interact hydrophobically with the macromolecule of serum albumin. The stability of supracomplex is dependent on pH value, i.e. on the protonation of the pendant amino group of the complex (pKA = 5.6) and this interaction was confirmed from 1 H-NMRD profile and fluorescent analysis. The compound was tested for its angiographic properties in vivo on rat model. Furthermore, other complexes of the ligand with trivalent lanthanides (Nd3+ , Eu3+ , Tb3+ , Dy3+ , Er3+ ) were characterized by various methods (XRD, luminescence, UV-VIS, 1 H-, 17 O- and 31 P-NMR). The cleavage of the benzyl groups affords ligand whose Ln3+ complexes possess pH dependent PARACEST effect. These complexes were characterized by XRD, luminescence and 1 H- and 31 P-NMR. Moreover, the novel ligands with modified length of pendant...
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Binding of organic dyes to proteins. Applications in practical course in biochemistry.
Hynková, Anna ; Hudeček, Jiří (advisor) ; Koblihová, Jitka (referee)
In this submitted thesis the possibility of creating a new laboratory task for advanced practical courses in biochemistry, concerning the binding of a low-molecular compound (dye) to a protein macromolecule, was experimentally verified. First intention was to modify the existing task "Dialysis kinetics" with a binding of fluorescein to a protein. However, the experiments have proved, that fluorescence measurements of this kind are not reproducible in the conditions of practical courses laboratory and absorbance measurements have low sensitivity. Therefor a whole new task was created: "Binding of bromophenol blue to serum albumin", in which the stoichiometry of this binding is studied using so-called Job plot. After the optimization of procedure a laboratory task instructions were created, which are attached to this thesis. Keywords: fluorescein, bromophenol blue, serum albumin
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Binding of low-molecular weight ligands to serum albumin
Buša, Michal ; Hudeček, Jiří (advisor) ; Vaněk, Ondřej (referee)
In this Thesis, we studied the possibilities of an enlargement of the existing practical task in the Advanced practical course in biochemistry I, dealing with interaction of a dye (bromophenol blue) with serum albumin. Experimental protocol was proposed and tested, allowing the inclusion of Scatchard plot (in addition to the previously used Job plot). As part of the Thesis, the new protocol for students is presented, which may help them to understand the most used approaches for studying the stoichiometry of protein-ligand complexes. In Czech.
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ANALYSIS OF NON-ENZYMATIC POSTTRANSLATIONAL MODIFICATED (GLYCATED) ALBUMIN BY NANO-LC/MS/MS
Šťastná, Zdeňka ; Pataridis, Statis ; Sedláková, Pavla ; Mikšík, Ivan
Posttranslational modifications of proteins are important reactions, which significantly affect the function of proteins in the organism. In principle, they can be divided into enzymatic and non-enzymatic modifications. Non-enzymatic reactions include glycation (earlier called nonezymatic glycosylation), which plays an important role in the development of chronic complications of diabetes mellitus, uremia, in the process of aging and degeneration of the brain.This work deals with the study of glycated albumins (human serum albumin and bovine serum albumin). Methodologically we used nano-liquid chromatography coupled to Q-TOF mass spectrometer. In vitro modified proteins were cleavaged by trypsin and arising peptides were separated on C18 nano column with trap-column. Peptides and their modifications were analysed by high-resolution Q-TOF mass spectrometer MaXis with precision determination of mass below 2 ppm. We found some modifications of proteins. Besides well known carboxymethyllysine new ones were determined - create mass shift 78, 132 and 218. Origin of these modifications is discussed and possible structure is presented. All found modifications were allocated to the structure of proteins and reactivity to various oxo-compounds was also examined
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