Water-soluble polyol-methanofullerenes as mitochondria-targeted antioxidants: Mechanism of action.

The mechanism of an antioxidant action of water-soluble polyol - methanofullerenes C60[C9H10O4(OH)4]6 and C60[C13H18O4(OH)4]6 as the mild uncouplers of an oxidative phosphorylation and respiration is postulated. According to this mechanism, hydroxyl group of methanofullerenols can be protonated under excess of protons in the intermembrane space of hyperpolarized mitochondria. Protonation of fullerene derivatives is confirmed by the decrease in their negative Zeta potential in the pH below 5.4. Heavily protonated methanofullerenols become positively charged and move into the mitochondrial matrix. As a consequence, the proton gradient is dissipated, which causes a decrease in mitochondrial transmembrane potential (ΔΨm) and reduction in ROS production.

Novel water-soluble methanofullerenes C60[C13H18O4(OH)4]6 and C60[C9H10O4(OH)4]6: Promising uncouplers of respiration and phosphorylation.

Here, we report for the first time on two novel water-soluble polyol-methanofullerenes which uncouple respiration and oxidative phosphorylation. A cytofluorimetric JC-1-based ratiometric assay was used to quantify mitochondrial potential Ψm in Yarrowia lipolytica cells exposed to the fullerenes tested. Both methanofullerenes significantly downregulated Ψm, thereby decreasing the subset of cells with high mitochondrial potential compared with intact control cells. The Ψm-low subset of Yarrowia lipolytica cells resulted from methanofullerenes exposure preserved physiological cell size and granularity patterns.

W-band transient EPR and photoinduced absorption on spin-labeled fullerene derivatives.

We investigated by W-band (94 GHz) transient electron paramagnetic resonance (TREPR) and photoinduced absorption (PIA) spectroscopy two fullerene derivatives bearing a nitroxide radical unit. After pulsed laser photoexcitation of the molecules in liquid toluene solution, complex EPR spectra are recorded, with lines in absorption and emission. The intrinsic higher spectral and temporal resolution of the W-band frequency leads to the assignment of all the lines in the spectrum and the determination of the sign and the absolute value of the exchange coupling between the fullerene in its photoexcited triplet state (S(T) = 1) and the radical (S(R) = 1/2). The two compounds with different fullerene-nitroxide spacers show opposite-ferromagnetic and antiferromagnetic-exchange couplings. The time evolution of the spectra and the polarization of the lines are interpreted in terms of several possible spin polarization mechanisms. The EPR measurements are complemented with PIA experiments.

Metal binding induces conversion of B- to the hybrid B-Z-form in natural DNA.

Highly polymerized herring testis DNA of the random nucleotide sequence has been studied in solution by circular dichroism and ultra-violet absorption spectrometry under various experimental conditions. At low temperature upon addition of 0.05 M NaCl or 1.15 M MgSO4 the DNA formed a helix that belonged to the B-family. As the temperature was increased a transition from the pure B- to the hybrid B-Z-form occurred in the presence of 1.15 M MgSO4. This transition occurred over a large range of temperatures and corresponded to a non-cooperative conformational change. A similar DNA transition was induced with 0.098 mM Co(NH3)6Cl3. However, in the presence of 5.3 M NaCl the DNA conformation was not similar to that observed in 1.15 M MgSO4 or 0.098 mM Co(NH3)6Cl3 independently on the environmental temperature. In 5.3 M NaCl the DNA is thought to undergo a transition from one to another right-handed conformation that could be intermediate partially dehydrated conformer arising on the first step in the sequential transition to the dehydration of the polynucleotide. Our results show that a realistic model of native DNA, bearing Z-tracts embedded in B-helixes, can be obtained upon binding of alkaline earth or transition metals.

Synthesis, structure and biological activity of nitroxide malonate methanofullerenes.

Two nitroxide methanofullerenes was synthesized for the first time, and their structures and biological activities studied. It was shown by X-ray single crystal analysis that the methanofullerene with two nitroxide groups forms a 1 : 2 inclusion complex with chloroform and has a nearly tetrahedral (diamond-like) arrangement of fullerene-fullerene interactions in the crystal. For the first time, it has been found that malonate nitroxide methanofullerene in combination with the known anticancer drug cyclophosphamide (CPA) shows high antitumor activity against leukemia P-388.

First observation of the hyperfine structure of an excited quintet state in liquid solution.

After pulsed photoexcitation of a new fullerene-linked bisnitroxide, a well resolved transient EPR spectrum is detected which is assigned to an excited quintet spin state generated by spin coupling of the nitroxides and the fullerene excited triplet.

Action of hexaamminecobalt on the activity of Serratia marcescens nuclease.

Using CD spectroscopic and kinetic analysis, a refined mechanism of Co(NH3)6(3+) action on activity of Serratia marcescens nuclease was elucidated. The mechanism was identical with previously found mechanisms of Mg2+ and C7H5O2Hg+. Similarly to Mg2+ and C7H5O2Hg+, Co(NH3)6(3+) binding to the DNA substrate induced changes in the secondary structure which resulted in changes of the enzymatic activity of the S. marcescens nuclease. Upon binding of 0.03 Co(NH3)6(3+) per DNA phosphate, highly polymerized DNA displayed A-form characteristics. The DNA transition from B-form to A-form intermediate was followed by a decrease of the nuclease activity. The diminishing nuclease activity was consistent with diminishing values of Km and Kcat. Co(NH3)6(3+) binding to the highly polymerized DNA caused a 1.7-2.8-fold decrease in Km, and 13.3-19.9 decrease in Vmax compared with Mg-DNA complex. A vast excess of Co(NH3)6(3+) did not affect the activity of S. marcescens nuclease if the DNA in the assay mixture remained in its B-form conformation. Preincubation of S. marcescens nuclease with Co(NH3)6(3+) did not influence the tertiary structure of the enzyme.