@phdthesis{
author = "Korać Jačić, Jelena K.",
year = "2020",
abstract = "Epinefrin je kateholamin sa značajnom fiziološkom ulogom. Konformacija ovog molekulautiče na njegovu interakciju sa drugim molekulima i na njegove biološke efekte. Na fiziološkimpH vrednostima, koordinativne sposobnosti epinefrina prema gvožđu i redoks interakcije epinefrinasa gvožđem su od suštinske važnosti za razumevanje dve veoma različite pojave. Prva pojava ještetno dejstvo koje hronični psihološki/sredinski stres izaziva na nivou kardiovaskularnog sistema.Druga pojava je umrežavanje kateholaminima bogatih biopolimera i struktura. Kako bi se rasvetlileuloge rastvarača i vodoničnih veza u interakcijama epinefrina sa gvožđem, proučavana jekonformacija epinefrina u vodi i polarnom rastvaraču dimetil sulfoksidu (DMSO).U ovoj disertaciji su predstavljeni rezultati proučavanja mehanizama interakcije epinefrinasa Fe2+ i Fe3+ jonima pri različitim koncentracionim odnosima na pH 7,4, odnosno na pH vrednostikoja odgovara fiziološkim uslovima. U svrhu istraživanja bioloških efekata epinefrina, u ovojdisertaciji su predstavljena ispitivanja efekta Epi-Fe3+ kompleksa na ćelije koje konstitutivnoeksprimiraju adrenergičke receptore.Konformacije epinefrina u polarnim rastvaračima, vodi i DMSO, su proučavane metodamaH nuklearne magnetne rezonance (1H NMR) kao i dvodimenzionalnim metodama nuklearnemagnetne rezonance, i to: 1H - 1H COSY, 1H - 15N HSQC i NOESY. Na NH2 i CH2 grupamaepinefrina su uočeni hemijski neekvivalentni protoni prilikom korišćenja DMSO kao rastvarača.Ove pojave nisu uočene kada je rastvarač bila voda. Analizom uticaja korišćenog rastvarača naNMR spektar, i analizom uticaja povećanja temperature uzorka na NMR spektar, dolazi se dozaključka da jedan od protona amino grupe formira jaku intramolekulsku vezu sa alifatičnomhidroksilnom grupom, koja je pak H donor drugoj intermolekularnoj vezi sa DMSO. PomoćuNOESY metode su prikupljeni podaci o prostornoj poziciji protona u bočnom lancu. Na taj način jeformiran 3D model konformacije epinefrina u DMSO. Ukratko, epinefrin formira dodatni petočlaniprsten koji sadrži bifurkovane intramolekulske/intermolekulske vodonične veze i zauzima strukturuoblika škorpiona, gde kateholni prsten predstavlja telo škorpiona, a bočni lanac predstavlja rep kojije povijen u smeru glave škorpiona. U vodi, kao rastvaraču, konformacija epinefrina ne posedujeintramolekulske vodonične veze pa je tada struktura ovog molekula najverovatnije definisanavodoničnim vezama sa molekulima vode.U okviru ove disertacije ispitivanjima je ustanovljeno da epinefrin sa Fe3+ jonima gradistabilne visokospinske komplekse čija je stehiometrija 1:1 ili 3:1. Stehiometrija ovog kompleksazavisi od odnosa koncentracija epinefrina i Fe3+ jona. Na kateholnom prstenu epinefrina atomikiseonika predstavljaju mesto za formiranje koordinacione veze unutar fiziološki relevantnogbidentatnog 1:1 kompleksa. Fe3+ katjon ima slab uticaj na redoks osobine epinefrina. Međutim,epinefrin i Fe2+ joni grade kompleks koji je jak redukcioni agens. Posledica je redukcija O2,proizvodnja vodonik peroksida i formiranje Epi-Fe3+ kompleksa. U ovom procesu epinefrin se neoksiduje, odnosno Fe2+ jon nije prenosilac, već donor elektrona. Oksidacija Fe2+ jona koja jekatalizovana epinefrinom predstavlja moguće hemijsko objašnjenje za stresom izazvana oštećenjaćelija srca. Takođe, rezultati ovih ispitivanja su u skladu sa prethodnim istraživanjima kateholaminau polimerima i njihovih interakcija sa gvožđem, i upućuju na nove strategije za poboljšavanjeefikasnosti umrežavanja kateholaminima bogatih biopolimera i struktura.U stresnim situacijama epinefrin se luči i može interagovati sa labilnim gvožđem koje senalazi u ljudskoj plazmi. Te interakcije mogu prouzrokovati značajne patofiziološke posledice. Uivovoj disertaciji su prikazani rezultati istraživanja prema kojima epinefrin i Fe3+ joni na fiziološkompH grade stabilni 1:1 bidentatni kompleks. Takođe je pokazano da na fiziološkom pH epinefrin nedegradira u prisustvu gvožđa. Utvrđeno je i da epinefrin i Fe2+ joni grade bezbojni kompleks i da jetaj kompleks stabilan pri anaerobnim uslovima. Uočeno je i da epinefrin u prisustvu O2 značajnopromoviše oksidaciju Fe2+ jona i formiranje Epi-Fe3+ kompleksa. Pri eksperimentima rađenimmetodom ciklične voltametrije, Epi-Fe2+ kompleks je pokazao potencijal E1/2 = -582 mV (u odnosuna standardnu vodoničnu elektrodu). Ovakva vrednost E1/2 potencijala objašnjava katalizovanuoksidaciju. Interakcije sa gvožđem mogu uticati na biološke efekte/efikasnost epinefrina. Uticajvezivanja gvožđa na biološko ponašanje epinefrina je ispitivan metodom nametnute voltaže nadeliću membrane u konfiguraciji cela ćelija, u kulturi ćelija koje konstitutivno eksprimirajuadrenergičke receptore. Epinefrin je samostalno, bez značajnog prisustva gvožđa, uzrokovaopovećanje amplitude struja usmerenih ka spoljašnosti ćelija, tj. povećanje izlaznih struja. Kompleksepinefrina sa Fe3+ nije izazivao slične posledice. Ovim se nameće zaključak da formiranjekompleksa sa gvožđem sprečava vezivanje epinefrina za adrenergičke receptore i njihovuposledičnu aktivaciju. Prooksidativna aktivnost Epi-Fe2+ kompleksa možda predstavlja vezu izmeđuhroničnog stresa i kardiovaskularnih problema, a labilno gvožđe u plazmi je potencijalni modulatorbioloških aktivnosti liganda., Epinephrine (Epi) is a catecholamine with important physiological roles. Interactions withother molecules and associated biological effects of Epi are controlled by its molecularconformation. Coordinate interactions of epinephrine with iron at physiological pH and their redoxactivity are crucial for understanding two distinct phenomena. First, the adverse effects that chronicstress causes to cardiovascular system. Second, the cross-linking of biopolymers and frameworkswhich are rich in catecholamines. Conformation of epinephrine in polar solvents, namely indimethyl sulfoxide (DMSO) and water, was investigated in order to shed light on effects solventsand hydrogen bonds exert on interactions of epinephrine with iron.Mechanism of epinephrine interactions with Fe2+ and Fe3+ ions was studied at differentconcentration ratios, at physiological pH (pH 7.4), and the results are presented in this dissertation.For the sake of exploration of biological effects of epinephrine, this dissertation also contains theresults of examination of effects Epi-Fe3+ complex has on cell culture with constitutive expressionof adrenergic receptors.Conformation of epinephrine in polar solvents, namely in dimethyl sulfoxide (DMSO) andwater, was investigated using 1H NMR, 1H - 1H COSY, NOESY and 1H - 15N HSQC methods.When DMSO was used as a solvent, chemical and magnetic nonequivalence of protons was spottedat NH2 and CH2 groups on epinephrine. Characteristics of hydrogen bonds in DMSO weredetermined by studying effect which temperature rise has on NMR spectra and also analyzinginfluences of solvent substitution on NMR spectra. Results have shown that epinephrine inducesstrong intramolecular bond between one of the protons of NH2 group and the OH group on the sidechain. On the other hand, the OH group on the side chain, i.e. the aliphatic OH group, presents aproton donor for intermolecular bond between epinephrine and DMSO. This phenomenon was notnoticed when water was used as a solvent. 3D modelling of epinephrine molecule structure wasbased on information about spatial arrangement of protons, which in turn was obtained usingNOESY method. Obtained 3D model shows that epinephrine in DMSO has a rigid structure thatresembles the shape of a scorpion, in which the catechol ring presents the body of the scorpion andthe side chain presents the tail of the scorpion. This structure is a consequence of formation of anadditional five–membered ring limited by inter/intra–molecular bonds. If water is used as a solvent(instead of DMSO), epinephrine takes different and non-rigid conformation which does not possessthe aforementioned intramolecular hydrogen bond. In this case, conformation of epinephrine isdetermined by hydrogen bonds with solvent molecules.Examinations conducted in the scope of this dissertation showed that epinephrine and Fe3+form stable high-spin complexes in 1:1 and 3:1 stoichiometry. Stoichiometry of these depends onconcentration ratio of epinephrine and Fe3+. Results acquired using Raman spectroscopy haveshown that 1:1 bidentate Epi–Fe3+ complex is formed by coordinative bonding of Fe3+ ions toepinephrine molecule through O atoms on the catechol ring. Effect of Fe3+ and Fe2+ ions on redoxproperties of epinephrine was studied using method of cyclic voltammetry. It was observed thatFe3+ ions do not significantly affect redox properties of epinephrine, but epinephrine with Fe2+ ionsforms strong reducing agent. Fe2+ ion presents electron donor that in the presence of epinephrinereduces O2 and causes production of H2O2. Specific hemism of epinephrine, which includesoxidation of Fe2+ ions, may present a mechanism that explains stress-induced cardiotoxicity andheart diseases. Also, these results can be used for improvement of synthesis and development ofbiopolymers.viIn stressful situations epinephrine is released and it may interact with labile iron in humanblood plasma. These interactions can have potentially important (patho)physiological effects. In thisdissertation, it is shown that at physiological pH epinephrine and Fe3+ build stable 1:1 high-spinbidentate complex. It is also shown that in presence of iron, at physiological pH, epinephrine doesnot degrade. It was observed that epinephrine and Fe2+ build colorless complex, which was stableunder anaerobic conditions. In presence of O2, epinephrine significantly catalyzed oxidation of Fe2+ions and formation of Epi-Fe3+ complex. Cyclic voltammetry results showed that the mid-pointpotential of Epi-Fe2+ complex equals -582 mV (vs. standard hydrogen electrode). This value ofmid-point potential explains the oxidation promotion. Biological effects/efficiency of epinephrineare influenced by its interaction with iron. Iron binding effects on biological performance ofepinephrine were examined using patch clamping in cell culture with constitutive expression ofadrenergic receptors. Epinephrine, on its own, induced an increase of outward currents, whereasEpi-Fe3+ complex did not evoke similar phenomenon. These imply that the binding of epinephrineto adrenergic receptors and their activation is inhibited by the formation of the complex of Epi withiron. Oxidative promoting activity of Fe2+ in the presence epinephrine may represent a basis forcardiovascular problems caused by chronic stress. The results obtained in this dissertation indicatethat the labile iron pool may have a new function that represents a modulation of the activity ofbiologically significant ligands/molecules.Epinephrine (Epi) is a catecholamine with important physiological roles. Interactions withother molecules and associated biological effects of Epi are controlled by its molecularconformation. Coordinate interactions of epinephrine with iron at physiological pH and their redoxactivity are crucial for understanding two distinct phenomena. First, the adverse effects that chronicstress causes to cardiovascular system. Second, the cross-linking of biopolymers and frameworkswhich are rich in catecholamines. Conformation of epinephrine in polar solvents, namely indimethyl sulfoxide (DMSO) and water, was investigated in order to shed light on effects solventsand hydrogen bonds exert on interactions of epinephrine with iron.Mechanism of epinephrine interactions with Fe2+ and Fe3+ ions was studied at differentconcentration ratios, at physiological pH (pH 7.4), and the results are presented in this dissertation.For the sake of exploration of biological effects of epinephrine, this dissertation also contains theresults of examination of effects Epi-Fe3+ complex has on cell culture with constitutive expressionof adrenergic receptors.Conformation of epinephrine in polar solvents, namely in dimethyl sulfoxide (DMSO) andwater, was investigated using 1H NMR, 1H - 1H COSY, NOESY and 1H - 15N HSQC methods.When DMSO was used as a solvent, chemical and magnetic nonequivalence of protons was spottedat NH2 and CH2 groups on epinephrine. Characteristics of hydrogen bonds in DMSO weredetermined by studying effect which temperature rise has on NMR spectra and also analyzinginfluences of solvent substitution on NMR spectra. Results have shown that epinephrine inducesstrong intramolecular bond between one of the protons of NH2 group and the OH group on the sidechain. On the other hand, the OH group on the side chain, i.e. the aliphatic OH group, presents aproton donor for intermolecular bond between epinephrine and DMSO. This phenomenon was notnoticed when water was used as a solvent. 3D modelling of epinephrine molecule structure wasbased on information about spatial arrangement of protons, which in turn was obtained usingNOESY method. Obtained 3D model shows that epinephrine in DMSO has a rigid structure thatresembles the shape of a scorpion, in which the catechol ring presents the body of the scorpion andthe side chain presents the tail of the scorpion. This structure is a consequence of formation of anadditional five–membered ring limited by inter/intra–molecular bonds. If water is used as a solvent(instead of DMSO), epinephrine takes different and non-rigid conformation which does not possessthe aforementioned intramolecular hydrogen bond. In this case, conformation of epinephrine isdetermined by hydrogen bonds with solvent molecules.Examinations conducted in the scope of this dissertation showed that epinephrine and Fe3+form stable high-spin complexes in 1:1 and 3:1 stoichiometry. Stoichiometry of these depends onconcentration ratio of epinephrine and Fe3+. Results acquired using Raman spectroscopy haveshown that 1:1 bidentate Epi–Fe3+ complex is formed by coordinative bonding of Fe3+ ions toepinephrine molecule through O atoms on the catechol ring. Effect of Fe3+ and Fe2+ ions on redoxproperties of epinephrine was studied using method of cyclic voltammetry. It was observed thatFe3+ ions do not significantly affect redox properties of epinephrine, but epinephrine with Fe2+ ionsforms strong reducing agent. Fe2+ ion presents electron donor that in the presence of epinephrinereduces O2 and causes production of H2O2. Specific hemism of epinephrine, which includesoxidation of Fe2+ ions, may present a mechanism that explains stress-induced cardiotoxicity andheart diseases. Also, these results can be used for improvement of synthesis and development ofbiopolymers.viIn stressful situations epinephrine is released and it may interact with labile iron in humanblood plasma. These interactions can have potentially important (patho)physiological effects. In thisdissertation, it is shown that at physiological pH epinephrine and Fe3+ build stable 1:1 high-spinbidentate complex. It is also shown that in presence of iron, at physiological pH, epinephrine doesnot degrade. It was observed that epinephrine and Fe2+ build colorless complex, which was stableunder anaerobic conditions. In presence of O2, epinephrine significantly catalyzed oxidation of Fe2+ions and formation of Epi-Fe3+ complex. Cyclic voltammetry results showed that the mid-pointpotential of Epi-Fe2+ complex equals -582 mV (vs. standard hydrogen electrode). This value ofmid-point potential explains the oxidation promotion. Biological effects/efficiency of epinephrineare influenced by its interaction with iron. Iron binding effects on biological performance ofepinephrine were examined using patch clamping in cell culture with constitutive expression ofadrenergic receptors. Epinephrine, on its own, induced an increase of outward currents, whereasEpi-Fe3+ complex did not evoke similar phenomenon. These imply that the binding of epinephrineto adrenergic receptors and their activation is inhibited by the formation of the complex of Epi withiron. Oxidative promoting activity of Fe2+ in the presence epinephrine may represent a basis forcardiovascular problems caused by chronic stress. The results obtained in this dissertation indicatethat the labile iron pool may have a new function that represents a modulation of the activity ofbiologically significant ligands/molecules.",
publisher = "Универзитет у Београду, Хемијски факултет",
journal = "Универзитет у Београду",
title = "Ispitivanje koordinativnih sposobnosti epinefrina prema Fe2+ i Fe3+ katjonima i redoks aktivnost nastalih kompleksa",
url = "https://hdl.handle.net/21.15107/rcub_nardus_17801"
}
