Nitroxide fluorescence analysis

TMDBIO for the alkyl radicals formed by initiator (hydroperoxide) thermolysis. The further quantitation of this reaction is difficult without assuming a kinetic model or obtaining further analytical information such as the actual concentration of nitroxide that has reacted during the retardation reaction. In the special case of the pro-fluorescent nitroxide, this can be achieved by fluorescence analysis of the formed alkoxyamine R2NOP. 

Rachel, K., Asuncionpunzalan, E. and London, E. (1995) Anchoring of tryptophan and tyrosine analogs at the hydrocarbon polar boundary in model membrane-vesicles - paralax analysis of fluorescence quenching induced by nitroxide-labelled phospholipids. Biochemistry 34,15475-15479. 

Similar to fluorescence depolarization and NMR, two limiting cases exist in which the molecular motion becomes too slow or too fast to further effect the ESR lineshape (Fig. 8) (35). At the fast motion limit, one can observe a narrow triplet centered around the average g value igxx + gyy + giz with a distance between lines of aiso = Axx- -Ayy- -A2,z)l3, where gu and Ajj are principal values of the g-tensor and the hyperflne splitting tensor A, respectively. At the slow motion limit, which is also referred to as the rigid limit, the spectrum (shown in Fig. 8) is a simple superposition of spectra for all possible spatial orientations of the nitroxide with no evidence of any motional effects. Between these limits, the analysis of the ESR lineshape and spectral simulations, which are based on the Stochastic Liouville Equation, provide ample information on lipid/protein dynamics and ordering in the membrane (36). 

A new photochrome-fluorescence-spin method for the simultaneous quantitative analysis of the redox status and viscosity of a medium has been developed [20], The method of the viscosity measurement is based on the use of double fluorescence-nitroxide molecules. 

Beside the estimation of medium microviscosity, quartz plates modified by BFLl were used for the quantitative analysis of ascorbate. For this purpose, a number of solutions of ascorbic acid were prepared with different concentrations, and the kinetics of change in steady-state fluorescence was recorded. There are two parallel processes that influence fluorescence (a) trans-cis photoisomerization and (b) reduction of the nitroxide moiety in traws-BFLl. After a correction taking into account the influence of trans-cis photoisomerization (curve b ), the pseudo-first-order kinetics of BFL reduction appears as curve c. The correction was done by performing a parallel measurement under identical conditions but without adding ascorbate. 

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