АННОТАЦИЯ
Евстигнеев М.П. Физические механизмы ассоциации биологически активных ароматических молекул и их конкурентного связывания с ДНК. –Рукопись.
Диссертация на соискание ученой степени кандидата физико-математических наук по специальности 03.00.02 – биофизика. – Харьковский национальный университет им. В.Н.Каразина, г.Харьков, 2002.
С целью установления молекулярных механизмов совместного связывания биологически активных ароматических лигандов с ДНК предложены методики анализа многокомпонентного равновесия в растворе, включающего процессы само- и гетероассоциации ароматических молекул и их комплексообразования с ДНК. Разработаны модели гетероассоциации ароматических молекул и их комплексообразования с ДНК в водном растворе для интерпретации данных ЯМР спектроскопии. Методом двумерной гомоядерной 1H-ЯМР спектроскопии (500 МГц) исследованы самоассоциация антибиотика новатрона и гетероассоциация акридиновых (профлавин и акридиновый оранжевый) и фенантридиновых (бромистый этидий и йодистый пропидий) красителей с антрациклиновым антибиотиком дауномицином. Определены равновесные константы и термодинамические параметры реакций ассоциации взаимодействующих молекул, построены пространственные структуры 1:1 комплексов. Сформулированы общие закономерности процессов ассоциации ароматических молекул в растворе.
Ключевые слова: ассоциация, статистико-термодинамическая модель, термодинамические параметры, ЯМР-спектроскопия, новатрон, дауномицин, ароматические красители, конкурентное связывание, ДНК.
SUMMARY
Evstigneev M.P. Physical mechanisms of association of biologically active aromatic molecules and their competitive binding with DNA. – Manuscript.
Thesis for a Candidate’s degree of Physical and Mathematical Sciences in Biophysics - Speciality 03.00.02, V.N.Karazin Kharkov National University, Kharkov, 2002.
NMR spectroscopy has been used to elucidate the molecular basis of a competitive binding with DNA of aromatic molecules, having different medical biological properties - mutagens and antibiotics. In order to analyse a multicomponent equilibrium in solution different interactions between molecules, including self- and hetero-association of aromatic ligands and their complexation with DNA have been studied.
Homonuclear one-dimensional and two-dimensional (2D-TOCSY and 2D-ROESY) 1H-NMR spectroscopy (500 MHz) has been used to investigate the self-association of antibiotic novatrone (NOV) and hetero-association of typical mutagens, acridine dyes (proflavine (PF) and acridine orange (AO)) and phenanthridine dyes (ethidium bromide (EB) and propidium iodide (PI)), with anthracycline antibiotic daunomycin (DAU) in aqueous solution.
Statistical-thermodynamical models of hetero-association of aromatic molecules have been developed for interpretation of NMR data, where there are no limitations on the magnitudes of the equilibrium self-association constants and concentrations of interacting molecules in aqueous solution. Comparison of the models has been made and limitations of their application have been formulated. On the basis of theoretical approaches a general algorithm of the calculation procedure using NMR data has been suggested. The method leads to determination of structures and thermodynamics of association of ligands in aqueous solution. Equilibrium constants and thermodynamical parameters (enthalpy and entropy) of the association reactions have been determined and the most favourable structures of 1:1 complexes have been calculated for all the systems studied.
It is concluded that 1:1 complexes of aromatic molecules in aqueous solution are stabilized by dispersive van der Waals, hydrophobic and H-bond interactions; the major role play dispersive interactions giving negative enthalpy-entropy change. Hydrophobic interactions have a substantial contribution in stabilization of NOV dimer and AO+DAU hetero-complex due to the existence of bulky side chains in the chromophores of NOV and AO molecules. Comparative analysis of the obtained association parameters shows relatively high magnitudes of equilibrium association constants and enthalpy/entropy change for NOV self-association and for hetero-association of mutagens (PF,EB,PI) with DAU. Taking into consideration intermolecular ROE cross-peaks and distances between donor and acceptor groups in 1:1 hetero-complexes of the interacting molecules, the obtained results are consistent with hydrogen bond formation between 3,6-aminogroups of PF and 9-MeCO group of DAU and also between EB/PI 3,8-aminogroups and 9-MeCO group of antibiotic.
Results of investigation of mutagen/antibiotic hetero-association were used for elucidation of the molecular mechanism of the binding of biologically active molecules with DNA in the mixed solution. An NMR derived model of complexation of aromatic ligands with DNA has been further developed to include no limitation on the self-association constants of the intercalating molecules. Analysis of the dynamic equilibrium of two aromatic ligands and DNA in aqueous solution, which includes the most probable interactions between molecules has been proposed. It has been found that the efficacy of intercalative binding of different aromatic molecules with DNA depends on both the hetero-association of the ligands (interceptor action) and the competitive binding of the ligands with DNA (protector action). Relative importance of each process is determined by the concentrations of the molecular components in solution and also by relation between the equilibrium constants of hetero-association of ligands and their complexation with DNA. Quantitative analysis has shown that complexation of PF and EB with oligonucleotide in the presence of DAU is highly affected by hetero-association of aromatic ligands (interceptor action) due to an extra stabilization of hetero-complexes in aqueous solution by formation of intermolecular H-bonds. It is concluded that such an analysis enables the optimum concentration of protector or interceptor molecules to be determined for any defined reduction in ligand binding to DNA.
Key words: association, statistical-thermodynamical model, thermodynamical parameters, NMR-spectroscopy, novatrone, daunomycin, aromatic dyes, competitive binding, DNA.
Наукове видання
ЄВСТИГНЄЄВ Максим Павлович
“Фізичні механізми асоціації біологічно активних ароматичних молекул та їхнього конкурентного зв’язування з ДНК”
Підписано до друку 15.02.2002 р. Формат 60x90 1/16
Друк офсетний. Умовн.-др.арк. 1.0.
Тираж 100 прим. Зам. №6
Видавництво “СевДТУ”, Севастополь, 53, Стрілецька бухта, Студмістечко, НМЦ
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