Oxidatív katalízis metalloenzim modellekkel = Oxidative catalysis using metalloenzyme models

Oxidatív katalízis metalloenzim modellekkel A bioszervetlen kémia egyik fontos kutatási irányzata a biomimetikus oxidációk vizsgálata. Munkánk során vizsgáltuk a pirokatechin oxidáz, fenoxazinon szintetáz, szuperoxid dizmutáz és kataláz enzimek funkcionális modellezését mangán, vas és nikkel komplexekkel. Kimutattuk, hogy a dioximáto-vas(II) és nikkel(II) komplexek gyorsítják pirokatechin származékok bázis katalizált oxidációját. A reakciók mechanizmusának tisztázása érdekében indokolt volt részletes reakciókinetikai mérések elvégzése. A ferroxim(II)komplex ([Fe(Hdmg)2(Melm)2] alkalmasnak bizonyult a fenoxazinon szintetáz modellezésére, 2-amino-fenol származékok, mint modell szubsztrátumok oxidációjában. Megállapítottuk, hogy a [Mn2(HL)2](BPh4)2 {ahol H2L egy ötfogú dioxim ligandum HON=C(CH3)C(CH3)=N-CH2CH2)2NH} szelektív katalizátorként viselkedik az adrenalin oxidációjában adrenokrommá karbonát pufferben. Kimutattuk, hogy a dioximáto-mangán(II)komplex gyorsítja a hidrogén peroxid bomlását vízzé és oxigénné, így a kataláz enzim funkcionális modelljének tekinthető. | Oxidative catalysis using metalloenzyme models One of the major subjects of bioinorganic chemistry is the study of biomimetic oxidations. We have studied catechol oxidase, phenoxazinone synthase, superoxide dismutase and catalase enzymes using manganese, iron and nickel complexes as functional models. We have established that dioximato-iron(II) and nickel(II) complexes accelerate the base-catalyzed oxidation of catechol derivatives. For establishing the underlying mechanism, we have carried out detailed kinetic studies of the catalytic oxidation. Ferroxime(II) is a suitable model for phenoxazinone synthase in the oxidation of 2-amino-phenol derivatives as model substrates. The complex [Mn2(HL)2](BPh4)2 {where H2L is the quinquedentate dioxime ligand HON=C(CH3)C(CH3)=N-CH2CH2)2NH} was found to be a selective catalyst for the oxidation of epinephrine to adrenochrome in carbonate buffer. We have demonstrated that dioximato-manganese(II)complex accelerate the disproportionation of hydrogen-peroxide into water and dioxygen in buffered aqueous solution thus can be regarded as catalase model

Physicochemical characterization of artificial nanoerythrosomes derived from erythrocyte ghost membranes

Colloidal stabile nanoerythrosomes with 200 nm average diameter were formed from hemoglobin-free erythrocyte ghost membrane via sonication and membrane extrusion. The incorporation of extra lipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), added to the sonicated ghosts, caused significant changes in the thermotropic character of the original membranes. As a result of the increased DPPC ratio the chain melting of the hydrated DPPC system and the characteristic small angle X-ray scattering (SAXS) of the lipid bilayers appeared. Significant morphological changes were followed by transmission electron microscopy combined with freeze fracture method (FF-TEM). After the ultrasonic treatment the large entities of erythrocyte ghosts transformed into nearly spherical nanoerythrosomes with diameters between 100 and 300 nm and at the same time a great number of 10–30 nm large membrane proteins or protein clusters were dispersed in the aqueous medium. The infrared spectroscopy (FT-IR) pointed out, that the sonication did not cause changes in the secondary structures of the membrane proteins under our preparation conditions. About fivefold of extra lipid – compared to the lipid content of the original membrane – caused homogeneous dispersion of nanoerythrosomes however the shape of the vesicles was not uniform. After the addition of about tenfold of DPPC, monoform and monodisperse nanoerythrosomes became typical. The outer surfaces of these roughly spherical objects were frequently polygonal, consisting of a net of pentagons and hexagons

Characterization of extracellular vesicles by IR spectroscopy: fast and simple classification based on amide and C-H stretching vibrations

Extracellular vesicles isolated by differential centrifugation from Jurkat T-cell line were investigated by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). Amide and C-H stretching band intensity ratios calculated from IR bands, characteristic of protein and lipid components, proved to be distinctive for the different extracellular vesicle subpopulations. This proposed ’spectroscopic protein-to-lipid ratio’, combined with the outlined spectrum-analysis protocol is valid also for low sample concentrations (0.15-0.05 mg/ml total protein content) and can carry information about the presence of other non-vesicular formations such as aggregated proteins, lipoproteins and immune complexes. Detailed analysis of IR data reveals compositional changes of extracellular vesicles subpopulations: second derivative spectra suggest changes in protein composition from parent cell towards exosomes favoring proteins with -turns and unordered motifs at the expense of intermolecular -sheet structures. The IR-based protein-to-lipid assessment protocol was tested also for red blood cell derived microvesicles for which similar values were obtained. The potential applicability of this technique for fast and efficient characterization of vesicular components is high as the investigated samples require no further preparations and all the different molecular species can be determined in the same sample. The results indicate that ATR-FTIR measurements provide a simple and reproducible method for the screening of extracellular vesicle preparations. It is hoped that this sophisticated technique will have further impact in extracellular vesicle research

Mesterséges és biológiai eredetű vezikulák infravörös spektroszkópiai vizsgálata = Artificial and biological vesicles: characterization by infrared spectroscopy

Az infravörös (FTIR) spektroszkópia megbízható, elterjedt módszer modell-, illetve biológiai membránok jellemzésére; ennek ellenére az általa nyújtott információk nincsenek teljes mértékben kiaknázva. Teljes reflexiós infravörös spektroszkópiával (ATR-FTIR) mesterséges és biológiai eredetű vezikulákat vizsgáltunk: i) a gyógyászatban már alkalmazott úgynevezett pegilált, azaz PEG-lánccal stabilizált vezikulák esetében a PEG lánc szerkezeti változásait követtük (a C-O-C kötések trans-gauche aránya alapján) az összetétel függvényében; ii) új típusú stabilizált vezikulák kifejlesztése céljából a vezikulákat felépítő lipid alapelemek és a különböző adalékanyagok (pl. urzolsav, koleszterin) kölcsönhatásait értelmeztük; iii) vörösvértest ghost membránból és mesterséges lipidekből fizikai-kémiai eljárásokkal előállított úgynevezett nanoeritroszómák esetében a mesterséges lipid beépülését vizsgáltuk; iv) egy egyszerű, gyors módszert dolgoztunk ki sejt-eredetű (Jurkat) extracelluláris vezikulák izolálásának jellemzésére a fehérje/lipid arány IR spektroszkópiai meghatározásával

The supramolecular chemistry of gold and l-cysteine: Formation of photoluminescent, orange-emitting assemblies with multilayer structure

The protein mediated approach is a common method for the synthesis of photoluminescent gold quantum clusters (GQCs), where proteins, acting as reducing and stabilizing agents, react with gold salts through cysteine residues. For the better understanding of the phenomenon, the aqueous phase reaction of HAuCl_4 and L-cysteine has been investigated at the supramolecular level by various experimental techniques and molecular mechanics simulations. We have observed the formation of a novel photoluminescent product, (AuCys)_n^β, which shows emission in the orange region of the spectrum. Small- and wide-angle X-ray scattering (SWAXS) measurements have revealed the presence of nanosized lamellae, which have an internal multilayer superlattice structure with a characteristic periodic distance of 1.3 nm. Based on the results, the layers are built up by zigzag shaped (AuCys)_n polymer chains connected through aurophilic bonds. The aurophilic network is stabilized via salt bridges and hydrogen bonds, which are also responsible for the interlayer interactions. Here, the evolution of the multilayer structure has been monitored by the combined application of photoluminescence spectroscopy and time-resolved SAXS. It has been concluded that there is a strong correlation between the emission and the scattering intensity, which suggests that the two- and three-dimensional aggregation of the building blocks to form sheets and multilayers are simultaneous processes. Furthermore, we have revealed that the formation and behavior of (AuCys)_n^β show significant differences to that of Au-L-glutathione compounds desrcibed earlier despite the similarity of L-cysteine and L-glutathione. These results evidence that L-cysteine and gold species form building blocks that can be applied expansively in supramolecular and cluster chemistry

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles

Extracellular vesicles (EVs) are released during the storage of red blood cell (RBC) concentrates and might play adverse or beneficial roles throughout the utilization of blood products (transfusion). Knowledge of EV release associated factors and mechanism amends blood product management. In the present work the impact of storage time and medium (blood preserving additive vs isotonic phosphate buffer) on the composition, size, and concentration of EVs was studied using attenuated total reflection infrared (ATR-IR) spectroscopy, microfluidic resistive pulse sensing (MRPS) and freeze-fraction combined transmission electron micrography (FF-TEM). The spectroscopic protein-to-lipid ratio based on amide and the C–H stretching band intensity ratio indicated the formation of various vesicle subpopulations depending on storage conditions. After short storage, nanoparticles with high relative protein content were detected. Spectral analysis also suggested differences in lipid and protein composition, too. The fingerprint region (from 1300 to 1000 cm−1) of the IR spectra furnishes additional information about the biomolecular composition of RBC-derived EVs (REVs) such as adenosine triphosphate (ATP), lactose, glucose, and oxidized hemoglobin. The difference between the vesicle subpopulations reveals the complexity of the REV formation mechanism. IR spectroscopy, as a quick, cost-effective, and label-free technique provides valuable novel biochemical insight and might be used complementary to traditional omics approaches on EVs