Studies of structure-activity relationship of nitroxide free radicals and their precursors as modifiers against oxidative damage.

The protective effects of stable nitroxides, as well as their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or exposure to ionizing radiation. Cytotoxicity was evaluated by monitoring the viability of the cells assessed by the clonogenic assay. The compounds tested at fixed concentration varied in terms of ring size, oxidation state, and ring substituents. Electrochemical studies were carried out to measure the redox midpoint potentials. The studies show that in the case of protection against H2O2 exposure, the protection was determined by the ring size, oxidation state, and redox midpoint potentials. In general the protection factors followed the order nitroxides > hydroxylamines > amines. Both the six-membered ring nitroxides and substituted five-membered ring nitroxides were efficient protectors. For six-membered ring nitroxides, the compounds exhibiting the lowest midpoint potentials exhibited maximal protection. In the case of X-radiation, nitroxides were the most protective though some hydroxylamines were also efficient. The amines were in some cases found to sensitize the toxicity of aerobic radiation exposure. The protection observed by the nitroxides was not dependent on the ring size. However, the ring substituents had significant influence on the protection. Compounds containing a basic side chain were found to provide enhanced protection. The results in this study suggest that these compounds are novel antioxidants which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and identify structural determinants optimal for protection against individual types of damage.

Synthesis of alpha-aryl N-adamant-1-yl nitrones and using them for spin trapping of hydroxyl radicals.

alpha-Aryl N-adamant-1-yl nitrones were synthesized and evaluated with respect to the stability of the hydroxyl radical adduct. The polarity and water solubility of nitrones were altered with changing the alpha-aryl groups. Introduction of adamantane ring instead of tert-butyl group resulted in a reasonable good stability of hydroxyl radical adduct for biological measurements.

Myosin catalytic domain flexibility in MgADP.

Conventional EPR studies of muscle fibers labeled with a novel alpha-iodoketo spin label at Cys-707 of the myosin head revealed substantial internal domain reorganization on the addition of ADP to rigor fibers. The spin probes that are well-ordered in the rigor state become disordered and form two distinct populations. These orientational changes do not correspond to rotation of the myosin catalytic domain as a whole because other probes (maleimide and iodoacetamide nitroxides attached to the same Cys-707 of myosin head) report only a small (5-10 degrees) torsional rotation and little or no change in the tilt angle [Ajtai et al. (1992) Biochemistry 31, 207-17; Fajer (1994) Biophys. J. 66, 2039-50]. In the presence of ADP, the labeled domain becomes more flexible and executes large-amplitude microsecond motions, as measured by saturation-transfer EPR with rates (tau r = 150 microseconds) intermediate between the rotations of detached (tau r = 7 microseconds) and rigor heads (tau r = 2500 microseconds). This finding contrasts with an absence of global motion of the myosin head in ADP (tau r = 2200 microseconds) as reported by the maleimide spin label. Our results imply that the myosin head in a single chemical state (AM.ADP) is capable of attaining many internal configurations, some of which are dynamic. The presence of these slow structural fluctuations might be related to the slow release of the hydrolysis products of actomyosin ATPase.