Effects of ring size and polar functional groups on the glutathione peroxidase-like antioxidant activity of water-soluble cyclic selenides.

To elucidate the effects of ring structure and a substituent on the glutathione peroxidase- (GPx-) like antioxidant activities of aliphatic selenides, series of water-soluble cyclic selenides with variable ring size and polar functional groups were synthesized, and their antioxidant activities were evaluated by NADPH-coupled assay using H2O2 and glutathione (GSH) in water and also by NMR spectroscopy using H2O2 and dithiothreitol (DTT(red)) in methanol. Strong correlations were found among the GPx-like activity in water, the second-order rate constants for the oxidation of the selenides, and the HOMO energy levels calculated in water. The results support the conclusion that the oxidation process is the rate-determining step of the catalytic cycle. On the other hand, such correlations were not obtained for the activity observed in methanol. The optimal ring size was determined to be five. The type of substituent (NH2 < OH < CO2H) and the number can also control the activity, whereas the stereoconfiguration has only marginal effects on the activity in water. In methanol, however, the activity rank could not be explained by the simple scenarios applicable in water.

Fatty acid conjugates of water-soluble (±)-trans-Selenolane-3,4-diol: effects of alkyl chain length on the antioxidant capacity.

Fatty acid monoesters of the title compound (DHS(red) ), of variable carbon chain length (propionate, laurate, myristate, palmitate, and stearate), were synthesized, and their antioxidant capacities were evaluated by means of a lipid peroxidation assay with lecithin/cholesterol liposomes. The selenides with long alkyl chains exhibited significant antioxidant activity (IC50 =9-34 µM) against accumulation of lipid hydroperoxide. Incorporation of the myristate into the liposome was ≈50 % by EPMA analysis. Intermediacy of the selenoxide was examined by NMR. In addition, enhancement of interfacial redox catalytic activity was observed for the myristate, but not for PhSeSePh and edaravone, in a PhCl/H2 O biphasic peroxidation assay. These results suggested that a combination of a hydrophilic selenide moiety as a redox center with a long alkyl chain is an effective approach to selenium antioxidants with interfacial glutathione-peroxidase-like (GPx-like) activity. The activity can be controlled by the alkyl chain length.

Kinetic and thermodynamic analysis of the conformational folding process of SS-reduced bovine pancreatic ribonuclease A using a selenoxide reagent with high oxidizing ability.

Redox-coupled folding pathways of bovine pancreatic ribonuclease A (RNase A) with four intramolecular disulfide (SS) bonds comprise three phases: (I) SS formation to generate partially oxidized intermediate ensembles with no rigid folded structure; (II) SS rearrangement from the three SS intermediate ensemble (3S) to the des intermediates having three native SS linkages; (III) final oxidation of the last native SS linkage to generate native RNase A. We previously demonstrated that DHS(ox), a water-soluble selenoxide reagent for rapid and quantitative SS formation, allows clear separation of the three folding phases. In this study, the main conformational folding phase (phase II) has been extensively analyzed at pH 8.0 under a wide range of temperatures (5-45 °C), and thermodynamic and kinetic parameters for the four des intermediates were determined. The free-energy differences (ΔG) as a function of temperature suggested that the each SS linkage has different thermodynamic and kinetic roles in stability of the native structure. On the other hand, comparison of the rate constants and the activation energies for 3S â†’ des with those reported for the conformational folding of SS-intact RNase A suggested that unfolded des species (desU) having three native SS linkages but not yet being folded are involved in very small amounts (<1%) in the 3S intermediate ensemble and the desU species would gain the native-like structures via X-Pro isomerization like SS-intact RNase A. It was revealed that DHS(ox) is useful for kinetic and thermodynamic analysis of the conformational folding process on the oxidative folding pathways of SS-reduced proteins.

Characterization of kinetic and thermodynamic phases in the prefolding process of bovine pancreatic ribonuclease A coupled with fast SS formation and SS reshuffling.

In the redox-coupled oxidative folding of a protein having several SS bonds, two folding phases are usually observed, corresponding to SS formation (oxidation) with generation of weakly stabilized heterogeneous structures (a chain-entropy losing phase) and the subsequent intramolecular SS rearrangement to search for the native SS linkages (a conformational folding phase). By taking advantage of DHS(ox) as a highly strong and selective oxidant, the former SS formation phase was investigated in detail in the oxidative folding of RNase A. The folding intermediates obtained at 25 °C and pH 4.0 within 1 min (1S°-4S°) showed different profiles in the HPLC chromatograms from those of the intermediates obtained at pH 7.0 and 10.0 (1S-4S). However, upon prolonged incubation at pH 4.0 the profiles of 1S°-3S° transformed slowly to those similar to 1S-3S intermediate ensembles via intramolecular SS reshuffling, accompanying significant changes in the UV and fluorescence spectra but not in the CD spectrum. Similar conversion of the intermediates was observed by pH jump from 4.0 to 8.0, while the opposite conversion from 1S-4S was observed by addition of guanidine hydrochloride to the folding solution at pH 8.0. The results demonstrated that the preconformational folding phase coupled with SS formation can be divided into two distinct subphases, a kinetic (or stochastic) SS formation phase and a thermodynamic SS reshuffling phase. The transition from kinetically formed to thermodynamically stabilized SS intermediates would be induced by hydrophobic nucleation as well as generation of the native interactions.

One-electron redox processes in a cyclic selenide and a selenoxide: a pulse radiolysis study.

One-electron redox reactions of cyclic selenium compounds, DL-trans-3,4-dihydroxy-1-selenolane (DHS(red)), and DL-trans-3,4-dihydroxy-1-selenolane oxide (DHS(ox)) were carried out in aqueous solutions using nanosecond pulse radiolysis, and the resultant transients were detected by absorption spectroscopy. Both *OH radical and specific one-electron oxidant, Br(2)(*-) radical reacted with DHS(red) to form similar transients absorbing at 480 nm, which has been identified as a dimer radical cation (DHS(red))(2)(*+). Secondary electron transfer reactions of the (DHS(red))(2)(*+) were studied with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS(2-)) and superoxide (O(2)(*-)) radicals. The bimolecular rate constants for the electron transfer reaction between (DHS(red))(2)(*+) with ABTS(2-) was determined as 2.4 +/- 0.4 x 10(9) M(-1) s(-1). From this reaction, the yield of (DHS(red))(2)(*+) formed on reaction with *OH radical was estimated in the presence of varying phosphate concentrations. (DHS(red))(2)(*+) reacted with O(2)(*-) radical with a bimolecular rate constant of 2.7 +/- 0.1 x 10(9) M(-1) s(-1) at pH 7. From the same reaction, the positive charge on (DHS(red))(2)(*+) was confirmed by the kinetic salt effect. HPLC analysis of the products formed in the reaction of (DHS(red))(2)(*+) with O(2)(*-) radicals showed formation of the selenoxide, DHS(ox). In order to know if a similar mechanism operated during the reduction of DHS(ox), its reactions with e(aq)(-) were studied at pH 7. The rate constant for this reaction was determined as 5.6 +/- 0.9 x 10(9) M(-1) s(-1), and no transient absorption could be observed in the wavelength region from 280 to 700 nm. It is proposed that the radical anion (DHS(ox))(*-) formed by a one-electron reduction would get protonated to form a hydroxyl radical adduct, which in presence of proton donors, would undergo dehydration to form DHS(*+). Evidence for this mechanism was obtained by converting DHS(*+) to (DHS(red))(2)(*+) with the addition of DHS(red) to the same system. Quantum chemical calculations provided supporting evidence for some of the redox reactions.

Direct observation of conformational folding coupled with disulphide rearrangement by using a water-soluble selenoxide reagent--a case of oxidative regeneration of ribonuclease A under weakly basic conditions.

Oxidative regeneration pathways of bovine pancreatic ribonuclease A (RNase A), which has four SS linkages, were studied at 25 degrees C and pH 8.0 by using trans-3,4-dihydroxy-1-selenolane oxide (DHS(ox)), a new selenoxide reagent with strong oxidation power. The short-term folding study using a quench-flow instrument ( approximately 1 min) revealed that early intermediates (1S, 2S, 3S and 4S) are formed stochastically and irreversibly from the reduced protein (R) and do not have any stable structures. In the long-term folding study ( approximately 300 min), on the other hand, slow generation of the key intermediates (des[65-72] and des[40-95]) through SS rearrangement from the 3S intermediate ensemble was observed, followed by slight formation of native RNase A (N). The parallel UV and CD measurements demonstrated that formation of the key intermediates is accompanied with the formation of the native-like structures. Thus, DHS(ox) allowed facile identification of the conformational folding steps coupled with SS rearrangement on the major oxidative folding pathways.