Early stages of LDL oxidation: apolipoprotein B structural changes monitored by infrared spectroscopy.

Changes in the conformation of apoliprotein B-100 in the early stages of copper-mediated low density lipoprotein oxidation have been monitored by infrared spectroscopy. During the lag phase no variation in structure is observed, indicating that copper binding to the protein does not significantly affect its structure. In the propagation phase, while hydroperoxides are formed but the protein is not modified, no changes in secondary structure are observed, but the thermal profile of the band corresponding to alpha-helix is displaced in frequency, indicating changes in tertiary structure associated with this conformation but not with beta-sheet components. When aldehyde formation starts, a decrease of approximately 3% in the area of bands corresponding to alpha-helix and beta-sheet is produced, concomitantly with an increase in beta-turns and unordered structure. The two bands corresponding to beta-turns vary as well under these conditions, indicating changes in these structures. Also at this stage the thermal profile shows variations in frequency for the bands corresponding to both alpha-helix and beta-sheet.The results are consistent with the hypothesis that as soon as the polyunsaturated fatty acids from the particle core are modified, this change is reflected at the surface, in the alpha-helical components contacting the monolayer.Fil: Chehin, Rosana Nieves. Consejo Superior de Investigaciones Científicas; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad del País Vasco; EspañaFil: Rengel, David. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; EspañaFil: Milicua, José Carlos G.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Goñi, Félix M.. Universidad del País Vasco; España. Consejo Superior de Investigaciones Científicas; EspañaFil: Arrondo JL. Consejo Superior de Investigaciones Científicas; España. Universidad del País Vasco; EspañaFil: Pifat, Greta. Rudjer Bošković Institute; Croaci

Istraživanje vezanja kofeina na albumin iz ljudskoga seruma

Binding of caffeine to human serum albumin (HSA) was investigated with the aim of describing the binding parameters of the interaction. It was found that the results obtained by fluorescence spectroscopy are influenced by the non-negligible artifact, known as the inner filter effect due to the absorption of caffeine at the excitation wavelength (290 nm). Therefore, a suitable correction of the obtained data was performed and the binding constant for caffeine binding to HSA was estimated, revealing low affinity of caffeine for HSA Ks = (12 ±1) *10³ mol–1 dm. Further, electron paramagnetic resonance (EPR) spectroscopy, using three different positional isomers of spin labeled stearic acid, doxyl stearates, was applied to study the caffeine-HSA interaction in further detail. It was found that upon caffeine binding, the hyperfine splitting decreases for HSA labeled with 5-doxylstearate. This phenomenon may indicate either an increase in mobility or a local change in polarity sensed by reporter groups upon caffeine binding. These observations may be important for the functional characteristics of HSA.Predmet ovoga istraživanja je vezanje kofeina na albumin iz ljudskoga seruma (HSA) s ciljem određivanja točne metodologije obrade dobivenih eksperimentalnih podataka. Naime, ustanovljeno je da na rezultate iz literature dobivene uporabom fluorescencijske spektroskopije utječe učinak unutarnjega filtera, kojega nije moguć e zanemariti. Učinak unutarnjega filtera prisutan je uslijed snažne apsorpcije kofeina na valnoj duljini pobude fluorescencije HSA (290 nm). Stoga je u ovome radu provedena korekcija dobivenih eksperimentalnih podataka te je procijenjena konstanta vezanja kofeina na HSA. Nadalje, za istraživanje interakcije kofeina i HSA primijenjena je spektroskopija elektronske paramagnetske rezonancije uz uporabu triju izomera spinski označene stearinske kiseline, doksil-stearata. Dobiveni rezultati ukazuju na smanjenje hiperfinoga cijepanja spektra HSA obilježenoga 5-doksil-stearatom u prisutnosti kofeina. Taj rezultat, zajedno s uočenim promjenama spektralnih amplituda, upućuje na povećanje mobilnosti spinske oznake u prisutnosti kofeina, uslijed poveć anja lokalne pokretljivosti proteina

The Influence of Lower Alcohols on the Surface Lipid Monolayer in LDL

Interaction of methanol, ethanol, propanol and butanol with human plasma low density lipoproteins (LDL) was studied in this work. The surface lipid monolayer of LDL was spin labeled and the electron spin resonance (ESR) spectra were measured in the presence and absence of alcohols. The decomposition of the complex ESR spectra was performed via theoretical simulations of experimental data. The results gained from this study indicate that the influence of alcohol could be observed through the changes of lipid ordering in the surface of LDL monolayer. This observation supports the hypothesis on the mechanism of alcohol action through its interference with lipid-protein interactions at the level of macromolecular surface

Oxidation Induced Changes in Lipid Mobility in Lipoproteins Followed by Steady State Fluorescence Anisotropy

The oxidation induced changes in the lipid layer of the lipoprotein particles of LDL and HDLs have been followed by measuring the steady-state polarization anisotropy of the fluorescence probe TMA-DPH incorporated in the lipid layer. The mobility restriction gradually increases with oxidation in LDL and HDL2 indicating the oxidation-induced structuring of lipids, while the effect of oxidation in HDL3 is negligible

Probing Protein Stability with Non-natural Amino Acids

Quantitative replacement of methionine with its non-natural amino acid analogues, norleucine, selenomethionine and telluromethionine in human recombinant annexin V, is applied to study conformational and folding properties in solution. This procedure replaces each methionine sulphur atom with Se, Te or -CH2-, providing single-atom exchanges, or »atomic mutations«. Using guanidine chloride as denaturant, the estimated stabilities of protein variants are not significantly changed. The denaturation midpoints are shifted towards lower values owing to the increase in the hydrophobicity of exchanged residues. Co-operativity expressed in terms of m-values is also affected by such exchanges and is highly correlated with the physical properties of methionine and its analogues in solution. This approach can contribute to a detailed understanding of interactions of particular amino acids and their implications on protein folding and protein-ligand interactions

The ESR Kinetic Study of Lipid Phase in HDL

Two main high-density lipoprotein subfractions, HDL2 and HDL3, were spin labelled with TEMPO which partitions both in aqueous and lipid phase. The dynamics of the lipid phase was monitored ina the reduction of incorporated TEMPO with ascorbic acid. The reduction of the paramagnetic nitroxide into the nonparamagnetic hydroxilamine form decreases the ESR signal with time. The reduction curves show complex behaviour while the partition coefficients of TEMPO remain unchanged during the reaction. The reduction process in the samples containing HDL particles proceeds faster than in the aqueous solution of pure reactants, e.g. spin label and ascorbic acid. In order to explain experimental data the model for reduction of TEMPO by ascorbic acid is proposed. It assumes that the processes taking place in these heterogeneous systems are determined by the overall reaction rate, which depends on the local concentrations of the reactants, as well as on their transport properties in the particular phases

Oxygen Bonding to Haemoglobin. 17O NMR Spectrum A Second Look

It was found that the upper concentration limit in detecting the oxygen-17 NMR signal from ordinary solvent water in solutions of oxyhaemoglobin is about 12 · 10-3 M (in haem). From samples of oxyhaemoglobin prepared with oxygen gas enriched to 62° /o in 170 a signal could be detected within a few days in spite of higher protein concentrations. Such signals increased in couple of weeks up to three times. However, within this period of time the sealed, dissolved oxyhaemoglobin became deoxygenated showing the characteristic colour change, while the NMR 170 signal persisted. The pressure above such sealed solutions diminished and the mass-spectroscopic analyses showed two- to threefold enrichment in 170 of solvent water after the deoxy-conversion, while in the gas phase oxygen ¥,Tas replaced by carbon dioxide. There is no deoxy-conversion in samples prepared under sterile conditions or in presence of sodium azide (equimolar with haem). It is concluded that owing to bacterial contamination in haemoglobin solutions prepared under ordinary conditions oxygen from the gas-phase is being reduced into water through bacterial metabolism. Thus, the observed 170 NMR signals were due to the solvent water enriched in 170. A former paper* claiming detection of 170 NMR signal from oxygen bound to haemoglobin is thus invalidated