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

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

Low temperature electron-spin relaxation in the crystalline and glassy states of solid ethanol

X-band electron paramagnetic resonance (EPR) spectroscopy was used to study the spectral properties of a nitroxide spin probe in ethanol glass and crystalline ethanol, at 5 - 11.5 K. The different anisotropy of molecular packing in the two host matrices was evidenced by different rigid limit values for maximal hyperfine splitting in the signal of the spin probe. The significantly shorter phase memory time, , for the spin probe dissolved in crystalline ethanol, as compared to ethanol glass, was discussed in terms of contribution from spectral diffusion. The effect of low-frequency dynamics was manifested in the temperature dependence of and in the difference between the data measured at different spectral positions. This phenomenon was addressed within the framework of the slow-motional isotropic diffusion model [S. Lee, and S. Z. Tang, Phys. Rev. B 31, 1308 (1985)] predicting the spin probe dynamics within the millisecond range, at very low temperatures. The shorter spin-lattice relaxation time of the spin probe in ethanol glass was interpreted in terms of enhanced energy exchange between the spin system and the lattice in the glass matrix due to boson peak excitations.Comment: 16 pages, 4 figures, 36 reference

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

Electron spin-lattice relaxation in solid ethanol: the effect of nitroxyl radical hydrogen bonding and matrix disorder

The electron spin-lattice relaxation of TEMPO and TEMPONE was measured at temperatures between 5 and 80 K in crystalline and glassy ethanol using X-band electron paramagnetic resonance spectroscopy. The experimental data at the lowest temperatures studied were explained in terms of electron-nuclear dipolar interaction between the paramagnetic center and the localized excitations, whereas at higher temperatures low-frequency vibrational modes from the host matrix and Raman processes should be considered. The strong impact of hydrogen bonding between the dopant molecule and ethanol host on the spin relaxation was observed in ethanol glass whereas in crystalline ethanol both paramagnetic guest molecules behaved similarly.Comment: 13 pages, 2 figures, 32 reference

Conformational Studies in Solid State and Solution of Protected C-terminal Dipeptide Fragment (Boc-Phe-Pro-NH2) of Morphiceptin

The crystal structure of the protected C-terminal dipeptide fragment (Boc-Phe-Pro-NH2) of the μ-opioid receptor highly selective agonist, morphiceptin (Tyr-Pro-Phe-Pro-NH2), was determined; the crystals are monoclinic with space group P21 and unit cell dimensions: a = 11.5731(5), b = 6.4490(3), c = 15.4082(5) Å, β = 100.359(5)° and Z = 2. To examine the influence of proline on the conformation of peptide bond, the molecular conformation was studied in solid state and solution (using 1H and 13C NMR data). The X-ray analysis revealed the following conformations of peptide backbone: φ1 = -63.2(5)°, ψ1 = 156.1(4)°, ω1 =-174.3(4)°, φ2 =-66.0(5)° and ψ2 = 152.0(4)°. The conformation of the Boc group is trans-trans. Experimental data revealed the trans conformation about the Phe-Pro amide bond, both in solid state and solution (DMSO). The possibility of cis/trans isomerization about the peptide bond (ω1) was examined by theoretical calculations using BIOSYM software. Molecular modelling, including molecular dynamics simulations of the title dipeptide, is in favour of trans peptide bond