tempo and tempo-carboxylic-acid

The fluorescence quenching of 7-amino-4-methylcoumarin by different TEMPO derivatives was studied in aqueous solutions with the use of steady-state, time-resolved fluorescence spectroscopy as well as UV-VIS absorption spectroscopy methods. In order to distinguish each TEMPO derivative from the others and to understand the mechanism of quenching, the absorption and fluorescence emission spectra as well as decays of the fluorescence of 7-amino-4-methylcoumarin were registered as a function of each TEMPO derivative concentration. There were no deviations from a linearity in the Stern-Volmer plots (determined from both, steady-state and time-resolved measurements). The fluorescence quenching mechanism was found to be entirely collisional, what was additionally confirmed by the registration of Stern-Volmer plots at 5 temperatures ranging from 15 to 55°C. Based on theoretical calculations of molecular radii and ionization potentials of all TEMPO derivatives the mechanism of electron transfer was rejected. The fluorescence quenching which was being studied seems to be diffusion-limited and caused by the increase of non-radiative processes, such as an internal conversion and an intersystem crossing. The Stern-Volmer quenching constants and bimolecular quenching constants were determined at the room temperature for all TEMPO derivatives studied. Among all TEMPO derivatives studied TEMPO-4-amino-4-carboxylic acid (TOAC) was found to be the most effective quencher of 7-amino-4-methylcoumarin fluorescence (kq for TOAC was approximately 1.5 higher than kq for other TEMPO compounds studied). The findings demonstrate the possibility of developing an analytical method for the quantitative determination of TOAC, which incorporation into membrane proteins may provide a direct detection of peptide backbone dynamics.

Topics: Coumarins; Cyclic N-Oxides; Fluorescence; Kinetics; Spectrometry, Fluorescence

Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike the others, the apolar TEMPO radical does not enter the cavity but instead binds to ferritin, presumably at a hydrophobic region of the protein. The kinetic data indicate that diffusion is not purely passive, the driving force coming not only from the concentration gradient between the inside and outside of the protein but also from charge interactions between the diffusant and the protein. A model for diffusion is derived that describes the observed kinetics. First-order half-lives for diffusion into the protein of 21-26 min are observed, suggesting that reductant molecules with diameters considerably larger than approximately 9 A would probably enter the protein cavity too slowly to mobilize iron efficiently by direct interaction with the mineral core.

Topics: Animals; Binding Sites; Biophysical Phenomena; Biophysics; Cyclic N-Oxides; Diffusion; Electrochemistry; Electron Spin Resonance Spectroscopy; Ferritins; Free Radicals; Horses; Kinetics; Nitrogen Oxides; Spin Labels; Spleen

The diagnosis of various disorders of the cerebrospinal fluid (CSF) with magnetic resonance (MR) imaging may require the intrathecal administration of a paramagnetic contrast agent. Furthermore, the CSF route provides direct access to the brain, circumventing the blood-brain barrier. Three nitroxides, two charged and one uncharged, were administered intrathecally to dogs to assess their potential as contrast agents for MR imaging of the CSF. Nitroxide concentrations and proton T1 values were measured in samples of CSF removed at various times after nitroxide administration, and pharmacokinetic curves were constructed. The charged nitroxides had considerably longer half-lives than the uncharged compound. On in vivo MR imaging of the CSF (surrounding the upper cervical cord and brain stem) in one dog, use of a charged nitroxide as contrast agent led to considerably higher CSF signal intensity than was observed in the nonenhanced, baseline T1-weighted images. This effect was achieved at low doses (20 mumol) and sustained for at least 90 minutes. The intrathecal use of nitroxides as contrast agents for MR imaging warrants continued investigation.

Topics: Animals; Cerebrospinal Fluid; Contrast Media; Cyclic N-Oxides; Dogs; Half-Life; Injections, Spinal; Magnetic Resonance Spectroscopy; Male; Spin Labels