Dubravka Krilov

FT-IR spectroscopy of lipoproteins—A comparative study

FT-IR spectra, in the frequency region 4000-600 cm(-1), of four major lipoprotein classes: very low density lipoprotein (VLDL), low density lipoprotein (LDL) and two subclasses of high density lipoproteins (HDL(2) and HDL(3)) were analyzed to obtain their detailed spectral characterization. Information about the protein domain of particle was obtained from the analysis of amide I band. The procedure of decomposition and curve fitting of this band confirms the data already known about the secondary structure of two different apolipoproteins: apo A-I in HDL(2) and HDL(3) and apo B-100 in LDL and VLDL. For information about the lipid composition and packing of the particular lipoprotein the well expressed lipid bands in the spectra were analyzed. Characterization of spectral details in the FT-IR spectrum of natural lipoprotein is necessary to study the influence of external compounds on its structure.

An EPR study of the transfer and trapping of holes produced by radiation in guanine(thioguanine) hydrochloride single crystals.

Single crystals of guanine hydrochloride monohydrate, guanine hydrochloride dihydrate and anhydrous guanine dihydrochloride, doped with thioguanine, were irradiated with X and gamma rays. In all three systems the dominant radicals were associated with thioguanine. In the former two systems the stabilized species is the thiyl radical, formed by initial loss of an electron at some of the guanines in the crystal lattice, followed by hole migration to thioguanine and subsequent deprotonation of the radical formed. In the anhydrous guanine(thioguanine) dihydrochloride, that process is followed by acquisition of a chlorine ion. In the guanine hydrochloride monohydrate and guanine hydrochloride dihydrate lattices, systems of interacting closely spaced stacked bases and strings of chloride ions might support the migration of electrons and/or holes. In anhydrous guanine dihydrochloride, neither the bases nor the Cl- ions alone are capable of providing the means for the long-range electron, energy and spin transfer. It is the interchangeable sequence of the charged bases and the Cl- ions that makes the supporting strings or networks. The ultimate chlorination of the thioguanine-centered electron-loss radicals depends mainly on the availability of the Cl- ions and the space for their accommodation in the vicinity of the sulfur atom.

ESR evidence for the radiation-induced breakage of the sugar-phosphate bonds in nucleotides: single crystal of deoxycytidine 5'-monophosphate

Abstract Electron spin resonance has been used for the identification of the radicals in the gamma-irradiated single crystal of deoxycytidine 5′-monophosphate monohydrate. From the number and angular dependence of the proton couplings the =C(4′)—Ċ(5′)—H2(5′) radicals have been identified. These radicals are formed by the breakage of the sugar—phosphate bond.

Postirradiation Long-Range Energy Transfer in a Single Crystal of Cytosine Monohydrate: An EPR Study

Thiocytosine molecules incorporated in the cytosine monohydrate crystal lattice act as traps for both electrons and holes. The radiation-induced cytosine ion radicals, C(+) and C(-), release their charge upon heating. The excess electrons and holes migrate long distances in the crystal lattice. The migration of holes has been demonstrated by the postirradiation, thermally activated accumulation of thiocytosine cation radicals, T(+), and the migration of electrons by formation of the S-centered radicals of an anionic nature. It is estimated that the migration length of the holes is at least 30 interbase distances, and the migration length of the electrons is more than 100 interbase distances. The selective formation of the cationic and anionic trap radicals, depending on the trap concentration, is discussed in terms of differences between the migration of electrons and holes.

Radiation energy transfer and trapping in single crystals of hemihydrate and hydrochloride of 5-methylcytosine doped with 5-methylthiocytosine : An EPR study

Abstract Two different crystals of 5-methylcytosine with 5-methylthiocytosine as impuritiy, irradiated with ionising radiation, exhibit quite different types of paramagnetic centres associated with thio impurities. In 5-methylcytosine hemihydrate there are two types of radicals with the unpaired spin located mainly on sulphur. Both are of thiyl structure, presumably derived by a loss of an electron from 5-methylthiocytosine. In doped 5-methylcytosine hydrochloride two distinct paramagnetic species with high spin density on chlorine have been detected. One of them has been spectroscopically characterised and assigned to an electron-deficient species with the unpaired electron shared by a sulphur and a chlorine atom, with some delocalization over the pyrimidine ring. Both crystal lattices of 5-methylcytosine support migration of electrons/holes. In the hemihydrate lattice, the base stacking interaction, although weak in comparison to that in DNA or some other molecular crystals of the nucleic-acid bases, might be responsible for that. In the hydrochloride lattice, the chlorine-ion strings or layers might provide the means for the hole transport. Both neutral and positively charged 5-methylcytosine is a good radiation energy trap (hole trap).

ESR spectroscopy of the sulphur-centered radicals derived from thiocytosine

Abstract Cytosine: thiocytosine crystals are found to be suitable for the observation of radiation-energy transfer to the sulphur-containing species. At low temperatures the thiocytosine cations are formed by a transfer of excitations to thiocytosine in the host matrix. At elevated temperatures the thiocytosine protonated anions are observed. The latter radicals are formed by an electron transfer from the cytosine anion in a thermally activated process, with a subsequent protonation on S-2. The principal elements of the g tensors ( g xx =2.132, g yy =2.004 and g zz =2.002 for the cation and g xx =2.066, g yy =2.008 and g zz =2.000 for the protonated anion) are in agreement with the g values for other sulphur-centered radicals of similar structures.

ESR study of radiation‐induced radicals in the sugar‐phosphate region of nucleotides. III. The alkoxy radical in deoxycytidine 5′‐phosphate

The unstable radical at 77 K in a gamma‐irradiated single crystal of deoxycytidine 5′‐phosphate was analyzed by ESR. A very anisotropic g tensor (g1=1.9997, g2=2.0095, and g3=2.0773) and two approximately equal β‐proton couplings with isotropic values of 8.0 G were interpreted to represent a radical of the O–CH2–R form. The radical is formed by the breakage of the phosphate–ester bond. The calculated spin densities on H(5′) and H′(5′) in the INDO MO approximation are in agreement with the observed couplings. The lack of any hydrogen bonding in the radical is believed to bring about much smaller β‐proton couplings from those previously observed and assigned to the same radical.

Slow oxidation of high density lipoproteins as studied by EPR spectroscopy

There is relatively little information on the role of high density lipoprotein (HDL) oxidation in atherogenesis although there are indications that oxidation might affect atheroprotective activities of HDL. Recently we reported the study on LDL oxidation initiated and sustained by traces of the transition metal ions under conditions, which favor slow oxidation. Here we report the results of the analogous study on the oxidation of the two HDL subclasses. The oxidation process was monitored by measuring the time dependence of oxygen consumption and concentration of the spin-trapped free radicals using EPR spectroscopy. In both HDL2 and HDL3 subclasses, the dependence of the oxidation process on the copper/lipoprotein molar ratio is different from that in LDL dispersions. Comparison of the kinetic profiles of HDL2 and HDL3 oxidation revealed that under all studied experimental conditions HDL2 was more susceptible to copper-induced oxidation than HDL3.

Very slow autoxidation of low-density lipoprotein spares α-tocopherol

Abstract Low-density lipoprotein (LDL) in solution, supplemented with EDTA, is spontaneously oxidized at physiological temperature. In closed systems, three distinct phases of oxidation are present. In the lag period no change of any of the usually measured ‘markers’ of oxidation have been detected. In the second phase oxygen is consumed and subsequently the level of lipid hydroperoxides is increased. In the third phase, after consumption of oxygen, hydroperoxides decrease in concentration and the apoprotein-associated free radicals are formed. In the entire process α-tocopherol is conserved. The participation and preservation of α-tocopherol in the process is interpreted in terms of the tocopherol-mediated peroxidation. In the latter two phases α-tocopherol oscillates between the oxidized and reduced states.

Spectroscopic studies of alpha tocopherol interaction with a model liposome and its influence on oxidation dynamics

The influence of α-tocopherol on the surface conformation of liposome, as a model component of lipoproteins, and its role in oxidation process were studied. FT-IR spectra from suspensions of neat liposome, mixtures of liposome and α-tocopherol and liposome with incorporated α-tocopherol were analyzed. When α-tocopherol was incorporated into liposome, intensities of some bands were decreased or increased in comparison with the spectra of liposome and α-tocopherol mixture. These changes reflect the different localization of α-tocopherol in two types of liposome suspensions. The oxidation of liposome suspensions was initiated by addition of cupric ions. After prolonged oxidation, the differences in FT-IR spectra of oxidized samples were recorded. Differences were observed in comparison with spectra of native and oxidized liposomes were analyzed. The rate of oxidation was measured by EPR oximetry. Oxidation was generally very slow, but faster in liposome without α-tocopherol, indicating the protective role of α-tocopherol against liposome oxidation. On the other hand, liposome suspensions with EDTA in the buffer were not oxidized at all, while those with α-tocopherol and liposome mixture were only slightly oxidized. In this case the consumption of oxygen was the result of liposome oxidation supported by α-tocopherol. These results reflect the ambivalent role of α-tocopherol in liposome oxidation, similarly to findings in studies of lipoprotein oxidation.